First-in-class of shmt2 and mthfd2 inhibitors as antitumor agents

ABSTRACT

A compound of the Formula I and optionally a pharmaceutically acceptable salt thereof is provided:Formula I, wherein, R is one selected from the group consisting of H and CH3; n is an integer 4 when X is -CH2- and Ar is 1,4-phenyl, or n is an integer ranging from 1 to 4 when X is -CH2-and Ar is either 2&#39;-fluoro-1,4-phenyl or 2,5-thienyl, or n is an integer ranging from 1 to 4 when X is one selected from the group consisting of O, S, -NH-, -NHCHO-, -NHCOCH3-, and -NHCOCF3- and Ar is one selected from the group consisting of (a) 1,4-phenyl, (b) 2&#39;-fluoro-1,4-phenyl, and (c) 2,5-thienyl, or n is an integer 3 when X is -CH2-, R is CH3 and Ar is 1,4-phenyl.

CROSS REFERENCE TO RELATED APPLICATION

This non-provisional patent application claims the benefit of co-pendingU.S. Pat. Application Serial No. 17/002,912, filed on Aug. 26, 2020,which claims the benefit of U.S. Pat. Application Serial No. 16/118,007,filed Aug. 30, 2018, now U.S. Pat. No. 10,793,573, granted Oct. 6, 2020,which claims the benefit of U.S. Provisional Pat. Application Serial No.62/552,432, filed Aug. 31, 2017 (expired). The entire contents of U.S.Pat. Application Serial No. 17/002,912, U.S. Pat. Application Serial No16/118,007, and U.S. Provisional Pat. Application Serial No. 62/552,432,are incorporated by reference into this non-provisional patentapplication as if fully written herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos. R01CA166711, R01 CA152316, and R01 CA53535, and T32 CA009531 and F30CA228221 and P30 CA022453, awarded by the National Institutes of Health.The government has certain rights in the invention.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to novel compounds of Formulae I, II, andIII, as described herein, and pharmaceutically acceptable salts thereof,that are effective to inhibit human tumor cells, including lung cancerand pancreatic cancer cells. This invention also relates tocompositions, which include these novel compounds, for use in treating apatient with cancer including providing to the patient a therapeuticallyeffective dose of one or more of the compounds and /or compositions asdescribed herein to a patient.

2. Description of the Background Art

Serine hydroxymethyltransferase 2 (SHMT2) and 5, 10-methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) are critical enzymes inmitochondrial one-carbon metabolism. SHMT2 has recently been identifiedas an oncodriver and its inhibition, as determined by SHMT2 andknockouts MTHFD2, generates glycine auxotrophs. There are no knowninhibitors of SHMT2 or MTHFD2. Thus, there is a need in the art todevelop compounds that are inhibitors of SHMT2 or MTHFD2, as well asanticancer agents against cancers, such as, but not limited to, lungcancer and pancreatic cancer.

Metabolic reprogramming to support tumor progression has emerged as ahallmark of cancer (1). Of the many altered metabolic pathwaysassociated with the malignant phenotype, one-carbon (C1) metabolism isparticularly notable (2-4). C1 metabolism depends on an adequate supplyof tetrahydrofolate (THF) metabolites and generates critical purine,thymidylate, and glycine metabolites essential for cell proliferationand tumor progression (3-5). Thymidylate and purine nucleotides aresynthesized de novo in the cytosol (3-5), and C1 enzymes such asthymidylate synthase (TS) and the purine biosynthetic enzymesglycinamide ribonucleotide (GAR) formyltransferase (GARFTase) and5-aminoimidazole-4-carboxamide (AICA) ribonucleotide (AICAR)formyltransferase (AICARFTase) (respectively, the third and ninth stepsin de novo purine biosynthesis) are important therapeutic targets forcancer (6-8). Serine biosynthesis from glycine in the cytosol involvesserine hydroxymethyltransferase (SHMT) 1 and uses C1 units from5,10-methylene-THF (2-4).

Cytosolic and mitochondrial C1 metabolic pathways are interconnected byan exchange of serine, glycine and formate, see FIG. 1 . Extracellularfolates are transported into cells by the reduced folate carrier (RFC),proton-coupled folate transporter (PCFT) and folate receptors (FRs) (9,10). Whereas cytosolic folates are transported into mitochondria via themitochondrial folate transporter (MFT) (SLC25A32) (11, 12),mitochondrial folates do not exchange with those in the cytosol (13). Incancer cells, the 3-carbon of serine is the major source of C1 units,and in mitochondria, serine catabolic enzymes including SHMT2,5,10-methylene-THF dehydrogenase 2 (MTHFD2) and 10-formyl-THF synthetase(reverse) (MTHFD1L) generate glycine and C1 units (i.e., formate) tosustain C1-dependent nucleotide and amino acid biosynthesis in thecytosol, see FIG. 1 . 10-Formyl-THF is resynthesized from formate in thecytosol by the trifunctional enzyme MTHFD1. 10-Formyl-THF is utilizedfor purine nucleotide biosynthesis and can be converted to5,10-methylene-THF for TS and SHMT1.

Several studies have implicated mitochondrial C1 metabolism as criticalto the malignant phenotype (14-17). A study (16) of messenger RNAprofiles for over one thousand enzymes spanning nearly two thousandtumors across nineteen different cancer types identified SHMT2 andMTHFD2 among the top five most upregulated genes, highlighting the keyrole of mitochondrial C1 metabolism across a wide spectrum of cancers.Metabolomics analyses of 219 extracellular metabolites from the NCI-60cancer cell lines showed that glycine consumption and the glycinebiosynthetic pathway strongly correlated with cancer cell proliferation(14). These findings, combined with evidence of functional shortages ofamino acids (e.g., glycine) in tumors (18), suggested a therapeuticopportunity for SHMT2 targeting in cancer.

SHMT2 is induced by hypoxic stress in Myc-transformed cells (19) and iscritical to tumor cell survival in the hypoxic, nutrient-poor tumormicroenvironment (15, 19). SHMT2 (or MTHFD2) knockout (KO) cells areviable and tumorigenic (albeit with decreased growth rates) innutrient-rich conditions, as reversal of cytosolic SHMT1(serine→glycine) provides sufficient C1 units to sustain some level ofde novo nucleotide biosynthesis (20). However, SHMT1 only restores asmall fraction of the C1 pools in wild-type (WT) cells (20). Further,SHMT1 does not generate sufficient glycine for protein, nucleotide andglutathione biosynthesis, rendering both SHMT2 and MTHFD2 KO cells asglycine auxotrophs (4). This, in part, was the impetus for studies of aseries of pyranopyrazole compounds (e.g., SHIN 1) with dual SHMT1/SHMT2inhibition (21). While structurally unrelated to folates, thesecompounds bound to the folate binding site in SHMT2 and showed in vitroanti-tumor efficacy (particularly with B-cell cancers). However, theywere inactive in vivo, likely reflecting their poor metabolicstabilities (21).

SUMMARY OF THE INVENTION

The present invention satisfies the above described needs by providingnovel compounds to potently inhibit human tumor cells including lungcancer and pancreatic cancer cells. In addition, the novel analogsaccording to the invention target a novel oncodriver, serinehydroxymethyltransferase from the mitochondria (SHMT2) and methylenetetrahydrofolate dehydrogenase (MTHFD2). Since there are no knowninhibitors of SHMT2 or MTHFD2, these novel compounds of this inventionare first-in-class SHMT2 and/or MTHFD2 inhibitors useful in cancertreatment.

One embodiment of this invention provides a compound of Formula I, andoptionally a pharmaceutically acceptable salt thereof:

wherein, R is selected from the group consisting of H and CH₃; n is aninteger ranging from 1 to 4; X is selected from the group of -CH₂- , O,S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃-; and Ar is selected from thegroup of (a) 1,4-phenyl, (b) 2'-fluoro-1,4-phenyl, and (c) 2,5-thienyl.

In a preferred embodiment of this invention, a compound of Formula I,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n=2, Ar is 1,4-phenyl, R is H, and X is CH₂. This compound isfurther identified herein as AGF291.

In a preferred embodiment of this invention, a compound of Formula I,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n=3, Ar is 1,4-phenyl, R is H, and X is CH₂. This compound isfurther identified herein as AGF300.

In a preferred embodiment of this invention, a compound of Formula I,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n=4, Ar is 1,4-phenyl, R is CH₃, and X is CH₂. This compound isfurther identified herein as AGF307.

In a preferred embodiment of this invention, a compound of Formula I,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n=3, Ar is 1,4-phenyl, R is CH₃, and X is CH₂. This compound isfurther identified herein as AGF312.

In a preferred embodiment of this invention, a compound of Formula I,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n=3, Ar is 2,5-thienyl, R is H, and X is CH₂. This compound isfurther identified herein as AGF318.

In a preferred embodiment of this invention, a compound of Formula I,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n=4, Ar is 2,5-thienyl, R is H, and X is CH₂. This compound isfurther identified herein as AGF320.

In a preferred embodiment of this invention, a compound of Formula I,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n=2, Ar is 2,5-thienyl, R is H, and X is CH₂. This compound isfurther identified herein as AGF331.

In a preferred embodiment of this invention, a compound of Formula I,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n is selected from the group of integers of 1,3, and 4, R is H,X is CH₂, Ar is selected from the group of 1,4-phenyl and 2,5-thienyl.

In a preferred embodiment of this invention, a compound of Formula I,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n is selected from the group of integers of 1, 2, 3, and 4, R isCH₃, X is CH₂, Ar is selected from the group of 1,4-phenyl and2,5-thienyl.

In a preferred embodiment of this invention, a compound of Formula I,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n is selected from the group of integers of 2, 3, and 4, R is H,X is selected from the group of O, S, NH, NHCHO, NHCOCH₃, and NHCOCF₃ ,and Ar is selected from the group of (a) 1,4-phenyl, (b)2'-fluoro-1,4-phenyl, and (c) 2,5-thienyl.

Another embodiment of this invention provides a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof Formula I, and optionally a pharmaceutically acceptable salt thereof:

wherein, R is selected from the group consisting of H and CH₃; n is aninteger ranging from 1 to 4; X is selected from the group of -CH₂- , O,S, -NH-, -NHCHO-, -NHCOCH3-, and -NHCOCF₃-; and Ar is selected from thegroup of (a) 1,4-phenyl, (b) 2'-fluoro-1,4-phenyl, and (c) 2,5-thienyl.Another embodiment of this invention provides the pharmaceuticalcomposition having the compound of Formula I, as described herein,including a pharmaceutically acceptable carrier.

In another embodiment of this invention, a compound is provided ofFormula II, and optionally a pharmaceutically acceptable salt thereof:

Formula II, wherein, R is selected from the group consisting of H andCH₃; n is an integer ranging from 1 to 4; X is selected from the groupof -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃-.

In a preferred embodiment of this invention, a compound of Formula II,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n is 3, R is H, X is CH₂. This compound is identified herein asAGF287.

In a preferred embodiment of this invention, a compound of Formula II,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n is selected from the group of integers of 1, 2, and 4, R is H,and X is CH2.

In a preferred embodiment of this invention, a compound of Formula II,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n is selected from the group of integers of 1, 2, 3, and 4, R isCH₃, and X is CH₂.

In a preferred embodiment of this invention, a compound of Formula II,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n is selected from the group of integers of 1, 2, 3, and 4, R isH, and X is selected from the group of O, S, NH, NHCHO, NHCOCH₃, andNHCOCF₃.

Another embodiment of this invention provides a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof Formula II, and optionally a pharmaceutically acceptable saltthereof:

Formula II, wherein, R is selected from the group consisting of H andCH₃; n is an integer ranging from 1 to 4; X is selected from the groupof -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃-. A furtherembodiment of this invention provides a pharmaceutical compositionhaving a compound of Formula II, as described herein, and including apharmaceutically acceptable carrier.

Another embodiment of this invention provides a compound of Formula III,and optionally a pharmaceutically acceptable salt thereof:

Formula III, wherein, R is selected from the group consisting of H andCH₃; n is an integer ranging from 1 to 4; X is selected from the groupof CH and N; Y is selected from the group of CH and N; and Z is selectedfrom the group of -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃-.

In a preferred embodiment of this invention, a compound of Formula III,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n is 2, and R is H, and X is N, Y is either CH or N, and Z isselected from the group of CH₂, O, S, NH, NHCHO, NHCOCH₃, and NHCOCF₃.

In a preferred embodiment of this invention, a compound of Formula III,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n=2, R is H, Z is CH₂, X is N and Y is CH. An example of thiscompound is identified herein as AGF315.

In a preferred embodiment of this invention, a compound of Formula III,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n is 3, and R is H, and X is either CH or N, Y is either CH orN, and Z is selected from the group of CH₂, O, S, NH, NHCHO, NHCOCH₃,and NHCOCF₃.

In a preferred embodiment of this invention, a compound of Formula III,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n is 3, R is H, Z is CH₂, X is N and Y is CH. An example of thiscompound is identified herein as AGF317.

In a preferred embodiment of this invention, a compound of Formula III,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n is an integer selected from the group of 1, 2, 3, and 4, and Ris H, and X is N, Y is CH, and Z is selected from the group of CH₂, O,S, NH, NHCHO, NHCOCH₃, and NHCOCF₃.

In a preferred embodiment of this invention, a compound of Formula III,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n is an integer selected from the group of 1, 2, 3, and 4, and Ris H, and X is CH, Y is N, and Z is selected from the group of CH₂, O,S, NH, NHCHO, NHCOCH₃, and NHCOCF₃.

In a preferred embodiment of this invention, a compound of Formula III,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n is an integer selected from the group of 1, 2, 3, and 4, and Ris CH₃, and X is N, Y is CH, and Z is selected from the group of CH₂, O,S, NH, NHCHO, NHCOCH₃, and NHCOCF₃.

In a preferred embodiment of this invention, a compound of Formula III,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n is an integer selected from the group of 1, 2, 3, and 4, and Ris CH₃, and X is N, Y is CH, and Z is selected from the group of CH₂, O,S, NH, NHCHO, NHCOCH₃, and NHCOCF₃.

Another embodiment of this invention provides a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof Formula III, and optionally a pharmaceutically acceptable saltthereof:

Formula III, wherein, R is selected from the group consisting of H andCH₃; n is an integer ranging from 1 to 4; X is selected from the groupof CH and N; Y is selected from the group of CH and N; and Z is selectedfrom the group of -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃-.A further embodiment of this invention provides the pharmaceuticalcomposition having the compound of Formula III, as described herein, anda pharmaceutically acceptable carrier.

Another embodiment of this invention provides a method of treating apatient having cancer comprising administering to said patient atherapeutically effective amount of a compound of Formula I, andoptionally a pharmaceutically acceptable salt thereof:

Formula I, wherein, R is selected from the group consisting of H andCH₃; n is an integer ranging from 1 to 4; X is selected from the groupof -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃-; and Ar isselected from the group of (a) 1,4-phenyl, (b) 2'-fluoro-1,4-phenyl, and(c) 2,5-thienyl. A preferred embodiment of this invention provides amethod of treating a patient having cancer comprising administering atherapeutically effective amount of at least one compound of Formula Iselected from the group of AGF 291, AGF 299, AGF300, AGF307, AGF312,AGF318, AGF320, AGF323, AGF331, AGF 347, AGF 355, and AGF 359, andoptionally a pharmaceutically acceptable salt thereof, to said patient.

Another embodiment of this invention provides a method of treating apatient having cancer comprising administering a therapeuticallyeffective amount of a compound of Formula II, and optionally apharmaceutically acceptable salt thereof:

Formula II, wherein, R is selected from the group consisting of H andCH₃; n is an integer ranging from 1 to 4; and X is selected from thegroup of -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃-. Apreferred embodiment of this invention provides a method of treating apatient having cancer comprising administering a therapeuticallyeffective amount of a compound of Formula II that is AGF287, andoptionally a pharmaceutically acceptable salt thereof, to said patient.

Another embodiment of this invention provides a method of treating apatient having cancer comprising administering a therapeuticallyeffective amount of a compound of Formula III, and optionally apharmaceutically acceptable salt thereof:

Formula III, wherein, R is selected from the group consisting of H andCH₃; n is an integer ranging from 1 to 4; X is selected from the groupof CH and N; Y is selected from the group of CH and N; and Z is selectedfrom the group of -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃-.A preferred embodiment of this invention provides a method of treating apatient having cancer comprising administering a therapeuticallyeffective amount of at least one compound of Formula III selected fromthe group of AGF 315 and AGF 317, and optionally a pharmaceuticallyacceptable salt thereof, to said patient.

Another embodiment of this invention provides a method of targetingmitochondrial metabolism comprising administering to a cancer patient aneffective amount of at least one compound selected from the group ofFormula I, and optionally a pharmaceutically acceptable salt thereof, ofFormula II, and optionally a pharmaceutically acceptable salt thereof,and of Formula III, and optionally a pharmaceutically acceptable saltthereof:

Formula I, wherein, R is selected from the group consisting of H andCH₃; n is an integer ranging from 1 to 4; X is selected from the groupof -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃-; and Ar isselected from the group of (a) 1,4-phenyl, (b) 2'-fluoro-1,4-phenyl, and(c) 2,5-thienyl; and

Formula II, wherein, R is selected from the group consisting of H andCH₃; n is an integer ranging from 1 to 4; and X is selected from thegroup of -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃-; and

Formula III, wherein R is selected from the group consisting of H andCH₃; n is an integer ranging from 1 to 4; X is selected from the groupof CH and N; Y is selected from the group of CH and N; and Z is selectedfrom the group of -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃-.

Another embodiment of this invention provides a method of targetingSHMT2 and MTHFD2 comprising administering to a cancer patient aneffective amount of at least one compound selected from the group ofFormula I, and optionally a pharmaceutically acceptable salt thereof, ofFormula II, and optionally a pharmaceutically acceptable salt thereof,and of Formula III, and optionally a pharmaceutically acceptable saltthereof:

Formula I, wherein, R is selected from the group consisting of H andCH₃; n is an integer ranging from 1 to 4; X is selected from the groupof -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃- ; and Ar isselected from one of the group of (a) 1,4-phenyl, (b)2'-fluoro-1,4-phenyl, and (c) 2,5-thienyl; and

Formula II, wherein, R is selected from the group consisting of H andCH₃; n is an integer ranging from 1 to 4; and X is selected from thegroup of -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃-; and

Formula III, wherein R is selected from the group consisting of H andCH₃; n is an integer ranging from 1 to 4; X is selected from the groupof CH and N; Y is selected from the group of CH and N; and Z is selectedfrom the group of -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃-.

BRIEF DESCRIPTION OF THE DRAWINGS

A full description of the invention may be gained from the followingdescription of the preferred embodiments of the invention when read inconjunction with the accompanying drawings:

FIG. 1 shows C1 metabolism is compartmentalized in the cytosol and themitochondria. Folates (and folate analogs) enter the cell through plasmamembrane facilitated folate transporters, PCFT and RFC. Serinecatabolism in the mitochondria beginning with SHMT2 generates glycineand formate, the latter of which is required for downstream cytosolic denovo purine nucleotide biosynthesis (by GARFTase and AICARFTase) in its10-formyl-THF form and thymidylate biosynthesis following conversion to5,10-methyene-THF (5,10-CH₂- THF) by MTHFD1. SHMT1 catalyzes theconversion of glycine to serine in the cytosol. AICA is metabolized toAICAR (ZMP), the AICARFTase substrate which circumvents GARFTase. Thearrows in FIG. 1 denote the net flux of C1 metabolism, however, mostreactions in the serine/glycine cycle are reversible.

FIG. 2 shows the design of novel 5-substituted pyrrolo[3,2-d]pyrimidinebenzoyl and thienoyl compounds of this invention, namely, identified ascompounds AGF 291, AGF 300, AGF 299, AGF347, AGF 355, AGF 331, AGF 318,and AGF 320. In the table are summarized key structural features for thevarious compounds of the present invention, including the bridge lengthsidentified by “n” in FIG. 2 (3, 4, or 5 carbons), thienoyl or benzoylside-chains, and for the latter, the 2' ring substituent (F or H). N/Ais an abbreviation for and means not applicable.

FIG. 3 shows the in vitro antitumor efficacy and identification oftargeted pathways and enzymes by novel pyrrolopyrimidine compounds inH460 tumor cells. Dose-response curves are shown for AGF94, anestablished GARFTase inhibitor (27) and the novel compounds AGF291,AGF320, and AGF347 without additions, or in the presence of adenosine(60 µM), AICA (320 µM), thymidine (10 µM) and/or glycine (130 µM). Theresults are mean values +/-standard deviations for 3 biologicalreplicates.

FIGS. 4A, 4B, 4C, 4D, 4E, 4F, and 4G show the targeted metabolomicsanalysis to identify intracellular enzyme targets of AGF291, AGF320, andAGF347. FIG. 4A shows a schematic of serine isotope label scrambling anddTTP isotope analysis. Heavy (²H) atoms are represented by filled incircles with lighter shading representing the flux through the cytosolincluding TS (forming M+2 dTMP and dTTP, as shown) and darker shadingrepresenting the flux through the mitochondria beginning with SHMT2(forming M+1 dTTP). Most steps are reversible as indicated. Adapted from(20). FIG. 4B and FIG. 4C show total serine pools for HCT116 sublinesincluding drug-treated WT cells (FIG. 4B) and the corresponding serineisotope distributions (FIG. 4C). FIG. 4D and FIG. 4E show total GAR(FIG. 4D) and AICAR (FIG. 4E) pools in H460 sublines includingdrug-treated WT cells with and without treatment with 1 mM formate.FIGS. 4F and 4G show relative dTTP pools (FIG. 4F) and dTTP isotopedistributions (FIG. 4G) in H460 sublines including drug-treated WT cellswith and without 1 mM formate. For FIG. 4D, FIG. 4E and FIG. 4F, resultsfor drug-treated and SHMT2 KD cells were normalized to vehicle-treatedWT ± formate or NTC ± formate samples, as appropriate. All data are meanvalues +/- standard deviations of three technical replicates. #, p <0.10; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ^ or ^(v) are used inplace of * to specify a significant increase or decrease, respectively.Statistical comparisons were with vehicle-treated WT ± formate or NTC ±formate samples, as appropriate. “ns” means not significant.

FIG. 5 shows in vivo efficacies of compound of this invention identifiedas AGF291 and GEM toward the MIA PaCa-2 PaC xenografts. Female ICR SCIDmice (10 weeks old; 19 g average body weight) were implanted bilaterallywith human MIA PaCa-2 PaC tumors. Beginning on day 3 followingsubcutaneous implantation, the mice were dosed as follows: AGF291, Q6dx3at 7.75 mg/kg/inj, total dose 23.5 mg/kg; and GEM, Q4dx3 at 120mg/kg/inj, total dose 480 mg/kg). T/C values were 19% for AGF291 and 26%for GEM.

FIG. 6 shows in vivo efficacies of the compound of this invention AGF347and GEM (background art compound) toward the MIA PaCa-2 PaC xenografts.Female ICR SCID mice (10 weeks old; 19 g average body weight) wereimplanted bilaterally with human MIAPaCa-2 PaC tumors. Beginning on day3 following subcutaneous implantation, the mice were dosed as follows:AGF347, Q2dx8 at 15 mg/kg/inj, total dose 120 mg/kg; and GEM, Q4dx4 at120 mg/kg/inj, total dose 480 mg/kg). T/C values were 19% for AGF291 and26% for GEM. For AGF347, the T-C (1000 mg) was 54 days and 1/5 mice wasdisease-free at 122 days.

FIG. 7 shows several of the chemical structures of the compounds of thisinvention, namely, Formula I, Formula II, and Formula III.

FIG. 8 shows resulting data for 5N-substituted pyrrolo[3,2-d]pyrimidinecompounds, namely AGF291 and AGF287, of the present invention.

FIG. 9 shows the structures of the compounds of this invention namelyAGF291 and AGF287, and reference compound AGF94.

FIGS. 10A, 10B, 10C, 10D, and 10E show expression of PCFT in primary PaCspecimens. FIG. 10A shows transcript levels for human PCFT, FIG. 10Bshows transcript levels for RFC, and FIG. 10C shows transcript levelsfor FRα in 4 normal pancreas. 19 PaCspecimens (OriGene) and the TM00176PaC PDX were measured by qPCR and normalized β-actin transcripts, and incomparison to KB tumor cells. FIGS. 10D and 10E showimmunohistochemistry (IHC) staining of PCFT that was performed with 99primary PaC specimens and 4 normal pancreas tissues from a commercialTMA (US Biomax, Inc.). The TMA was incubated with affinity-purifiedPCFT-specific antibody or rabbit IgG, the slides developed,counterstained and mounted. The slides were scanned at 20× by an AperioImage Scanner (Aperio Technologies, Inc.) for microarray images. Thetotal intensity of antibody positive staining of each tissue core wascomputed and plotted as relative values. Median values are shown ascross bars. Representative images are shown in panel FIG. 10D.

FIG. 11 shows the results of growth inhibition of KB human tumor cellsby the compounds of the present invention and the results of protectionexperiments with nucleosides, 5-aminoimidazole-4-carboxamide andglycine. Growth inhibition was measured by a fluorescence-based assay(Cell TiterBlue™). Cells were cultured in folate-free (FF) RPMI 1640/10%dialyzedfetal bovine serum, and antibiotics, with 2 nM leucovorin.Results are expressed as nM IC50s (n=3-10; mean + SEM in parenthesis)relative to untreated control cells. For KB cells, results aresummarized for the protective effects of nucleoside additions includingadenosine (Ade) (60 µM) or thymidine (Thd) (10 µM), or5-aminoimidazole-4-carboxamide (AICA) (320 µM), or glycine (Gly) (120µM).

FIGS. 12A-12H show docked poses of 5-formyl-THF (A), AGF291 (B), AGF320(C), andAGF347 (D) in the human dimeric SHMT2 crystal structure (PDB:5V7I) (42); crystal structure of 5-formyl THF triglutamate (E) in rabbitSHMT1 (PDB: 1L53) (43) and docked poses of AGF291 (F), AGF320 (G), andAGF347 (H) in the rabbit SHMT1 structure. Molecular modeling wasperformed using the induced fit docking protocol of Maestro (44, 45).The docking scores for all the proposed analogs in SHMT2 and SHMT1 arein Table 1S. Panels A-D: For AGF347 (panel D; best SHMT2 docking scoreof the series (Table 1S), the pyrrolo[3,2-d]pyrimidine scaffold occupiesthe pocket lined by Leu166, Asn410, Gly170, and Arg425. The 2-NH₂ groupmakes H-bond (faded plum color) with the backbone CO of Pro167 and N1makes H-bond with Thr411 hydroxyl group and backbone NH. The four carbonalkyl chain linker substitution orients in the similar way as the linkerat C6 position of 5-formylTHF in SHMT1 crystal structure (E). The2-fluorophenyl ring is sandwiched between Tyr176 and Tyr105. The 2-flurosubstitution makes steric clash with the carbonyl oxygen of amide groupforcing the amide group perpendicular to the phenyl ring, which furtherforces the H-bond with the Tyr176 and Tyr105 hydroxyl groups. The α- andγ-COOH groups of the glutamyl side chain make salt bridge (blue colordotted line) with Lys181 and Lys103. These ionic interactions aremaintained by AGF291 and AGF320, along with the aromatic side chainsphenyl and thienoyl, respectively, resulting in pi-pi interaction (darkcolor dotted line) with Tyr176. Panels E-H. AGF320 shows the highestdocking score among all the proposed analogs for SHMT1 (Table 1S). Thepyrrolo[3,2-d]pyrimidine scaffold for AGF320 (panel G) sits in thepocket occupied by Leu121, Leu127, Gly125, His203, Arg363, Lys346, andAsn347. The 2-NH₂ group makes H-bonds (faded color line) with thebackbone CO of Leu121 and Gly125. The 4-oxo make water mediated H-bondwith Tyr65 and His203. The α-COOH makes H-bond with backbone of Leu357and γ-COOH makes salt-bridge (dark dotted line) with Lys134B.

FIG. 13 shows folate transporter transcript expression of human tumorcell lines by RT-PCR. Transcript levels for reduced folate carrier(RFC), proton-coupled folate transporter (PCFT) and folate receptor (FR)α in H460, HCT116 and MIA PaCa-2, and IGROV1 cells were measured byreal-time RT-PCR with results normalized to those of β-actin and GAPDH.The results represent three experimental replicates with individualtransporter expression within each replicate normalized to the averageof the corresponding transporter expression across all IGROV-1replicates. The results are presented as mean values +/- standarddeviations. Although H460, HCT116 and MIA PaCa-2 cells all expressabundant RFC and PCFT, FRα expression is negligible.

FIG. 14 shows in vitro antitumor efficacy and identification of targetedpathways and enzymes by novel 5-substituted pyrrolo[3,2-d]pyrimidinebenzoyl and thienoyl compounds of this invention in HCT116 and MIAPaCa-2 tumor cells. Dose-response curves are shown for AGF94, anestablished glycinamide ribonucleotide formyltransferase (GARFTase)inhibitor (46) and the 5-substituted pyrrolo[3,2-d]pyrimidine compoundsAGF291, AGF320, and AGF347 of this invention without additions, or inthe presence of adenosine (60 µM), 5-aminoimidazole-4-carboxamide (AICA)(320 µM), thymidine (10 µM) and/or glycine (120 µM). The experimentswere performed in complete folate-free RPMI1640, supplemented with 25 nMleucovorin without added glycine (see methods as described herein). Theresults are presented as mean values ± standard deviations for at leastthree biological replicates, with growth of cells treated with drug ±metabolite normalized to the growth of cells treated with vehicle (i.e.,DMSO) ± metabolite.

FIGS. 15A-15L show targeted metabolomics analysis to identifyintracellular enzyme targets of compounds of this invention AGF291,AGF320, and AGF347 in HCT116, H460, and MIA PaCa-2 cells. Cells wereincubated with drug or vehicle for 16 hours in glycine-replete,folate-free complete RPMI 1640, supplemented with 25 nM leucovorin(included unlabeled serine). The medium was replaced with media(including drug or vehicle) including 250 µM [2,3,3-²H]serine foranother 24 hours, after which metabolites were extracted for LC-MSanalysis. FIG. 15A and FIG. 15C, respectively, show total serine poolsand serine isotope distributions for H460 cells. FIGS. 15B and 15D,respectively, show MIA PaCa-2 cells. FIG. 15E and FIG. 15F,respectively, show GAR and AICAR accumulations in HCT116 cells, andFIGS. 15I and15J, respectively, show MIA PaCa-2 cells. FIGS. 15G and15H, respectively, show total dTTP pools and dTTP serine isotopedistributions for HCT116. FIGS. 15 and 15L, respectively, show MIAPaCa-2 cells. Results reflect three technical replicates. #, p < 0.10;*, p < 0.05; **, p < 0.01; ***, p < 0.001; ^ or ^(v) are used in placeof * to specify a significant increase or decrease respectively; ns =not significant. All statistical comparisons were made betweendrug-treated/knockout/knockdown samples and DMSO-treated WT/NTC samples.

FIG. 16 shows the Western blot confirming H460 SHMT2 knockdown andHCT116 SHMT2 knockout. Whole-cell lysates of wild-type (WT),non-targeted control shRNA-transduced (NTC), and clonal SHMT2 knockdown(SHMT2 KD) H460 cells, along with HCT116 WT and SHMT2 CRISPR/Cas9knockout (KO) cells were resolved on 10% polyacrylamide gel with SDS andprobed with monoclonal rabbit anti-SHMT2 antibody (#12762; CellSignaling Technology, Danvers, MA). The blot was stripped and reprobedwith mouse anti-β-actin antibody (Sigma-Aldrich) as a loading control.Experimental details are described herein. The blots were scanned withan Odyssey infrared imaging system (LICOR Biosciences). Densitometryanalysis (values given are SHMT2 band intensities normalized to β-actinband intensities) revealed SHMT2 protein expression of H460 SHMT2 KD tobe 5.3% of that of H460 NTC, and HCT116 SHMT2 KO expression to be <1% ofthat of HCT116 WT.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used herein, the term “patient” means members of the animal kingdom,including but not limited to, human beings.

As used herein, the term “effective amount” or “therapeuticallyeffective amount” refers to that amount of any of the present compounds,salts thereof, and/ or compositions required to bring about a desiredeffect in a patient. The desired effect will vary depending upon theillness or disease state being treated. For example, the desired effectmay be reducing the tumor size, destroying cancerous cells, and/orpreventing metastasis, any one of which may be the desired therapeuticresponse. On its most basic level, a therapeutically effective amount isthat amount of a substance needed to inhibit mitosis of a cancerouscell. As used herein, “tumor” refers to an abnormal growth of cells ortissues of the malignant type, unless otherwise specifically indicatedand does not include a benign type tissue. The “tumor” may be comprisedof at least one cell and/or tissue. The term “inhibits or inhibiting” asused herein means reducing growth/replication. As used herein, the term“cancer” refers to any type of cancer, including but not limited to lungcancer, pancreatic cancer, and the like.

As used herein, the term “glutamate” will be understood as representingboth the ester form (glutamate) and the acid form (glutamic acid).

The novel compounds and pharmaceutically acceptable salts thereofprovide for treatment of tumors, or other cancer cells, in cancerpatients. The types of cancer can vary widely and in certainembodiments, the novel compounds and pharmaceutically acceptable saltsthereof are particularly useful for example, in treating lung cancer andpancreatic cancer.

The present invention is further directed to methods of synthesizing thenovel compounds.

The present invention further relates to methods of using theabove-described novel compounds, and pharmaceutically acceptable saltsthereof, in therapeutically treating patients with cancer according to amethod, such as, including the steps of:

-   a) employing the above-described compound, or pharmaceutically    acceptable salt thereof;-   b) incorporating said compound in a suitable pharmaceutical carrier;    and-   c) administering a therapeutically effective amount of said compound    incorporated in said carrier to a patient.

As used herein, the term “therapeutically effective carrier” refers toany pharmaceutical carrier known in the art, absent compatibilityproblems with the novel compounds of the invention. Generally, carriersinclude for example but not limited to, physiologic saline and 5%dextrose in water.

As will be understood by one skilled in the art, a therapeuticallyeffective amount of said compound can be administered by any means knownin the art, including but not limited to, injection, parenterally,intravenously, intraperitoneally, orally or, where appropriate,topically.

It is well within the skill of one practicing in the art to determinewhat dosage, and the frequency of this dosage, which will constitute atherapeutically effective amount for each individual patient, dependingon the severity or progression of cancer or cancer cells and/or the typeof cancer. It is also within the skill of one practicing in the art toselect the most appropriate method of administering the compounds basedupon the needs of each patient.

The compounds disclosed in the present invention all can be generallydescribed as antifolates.

The novel compounds have been discovered, unexpectedly, to potentlyinhibit human tumor cells including lung cancer and pancreatic cancercells. More particularly, the compounds and the pharmaceuticallyacceptable salts thereof used to treat pancreatic cancer are completelyunprecedented, as no other known antifolate compound that inhibitspancreatic cancer cells has been described in the background art.

Protection studies of metabolic end products, e.g., nucleosides andamino acids, with tumor cells indicate that these analogs unexpectedlytarget a novel oncodriver, serine hydroxymethyltransferase (SHMT2)and/or methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) from themitochondria. Typically, the inhibitory effects of antifolate compoundsare protected by adenosine or thymidine, required for DNA synthesis.However, these novel analogs, unexpectedly, require glycine along withadenosine for complete protection, clearly establishing thatmitochondrion one-carbon metabolism is being targeted.

It has been shown that metabolism of serine (the substrate for SHMT2)and biosynthesis using a one carbon unit derived from serine is potentlyinhibited in tumor cells treated with the novel compounds of the presentinvention, which provides further evidence that mitochondrialmetabolism, in general, and SHMT2 and/or MTHFD2, in particular, arebeing targeted.

Thus, the novel compounds of the invention, e.g., 5-substitutedpyrrolo[2,3-d]- and pyrrolo[3,2-d]pyrimidines, are first-in-classinhibitors of SHMT2 and/or MTHFD2, and are anticancer agents againstcancer, such as, but not limited to, lung cancer and pancreatic cancer.

One embodiment of this invention provides a compound of Formula I, andoptionally a pharmaceutically acceptable salt thereof:

Formula I, wherein, R is one selected from the group consisting of H andCH₃; n is an integer ranging from 1 to 4; X is selected from one of thegroup consisting of -CH₂-, O, S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃-; and Ar is selected from one of the group consisting of (a) 1,4-phenyl,(b) 2'-fluoro-1,4-phenyl, and (c) 2,5-thienyl. Those persons skilled inthe art will appreciate that any of the combination(s) of the moietiesidentified for the groups of R, X, and Ar, and integers identified forn, are embodiments of the present invention for any of the formula(ae)presented for Formula 1.

In a preferred embodiment of this invention, a compound of Formula I,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n=2, Ar is 1,4-phenyl, R is H, and X is CH₂. This compound isfurther identified herein as AGF291.

In a preferred embodiment of this invention, a compound of Formula I,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n=3, Ar is 1,4-phenyl, R is H, and X is CH₂. This compound isfurther identified herein as AGF300.

In a preferred embodiment of this invention, a compound of Formula I,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n=4, Ar is 1,4-phenyl, R is CH₃, and X is CH₂. This compound isfurther identified herein as AGF307.

In a preferred embodiment of this invention, a compound of Formula I,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n=3, Ar is 1,4-phenyl, R is CH₃, and X is CH₂. This compound isfurther identified herein as AGF312.

In a preferred embodiment of this invention, a compound of Formula I,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n=3, Ar is 2,5-thienyl, R is H, and X is CH₂. This compound isfurther identified herein as AGF318.

In a preferred embodiment of this invention, a compound of Formula I,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n=4, Ar is 2,5-thienyl, R is H, and X is CH₂. This compound isfurther identified herein as AGF320.

In a preferred embodiment of this invention, a compound of Formula I,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n=3, Ar is 1,4-phenyl, R is H, and X is O . This compound isfurther identified herein as AGF323.

In a preferred embodiment of this invention, a compound of Formula I,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n=2, Ar is 2,5-thienyl, R is H, and X is CH₂. This compound isfurther identified herein as AGF331.

In a preferred embodiment of this invention, a compound of Formula I,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n=4 , Ar is 1,4-phenyl, R is H, and X is CH₂. This compound isfurther identified herein as AGF299.

In a preferred embodiment of this invention, a compound of Formula I,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n= 2, Ar is 2'-fluoro-1,4-phenyl, R is H, and X is CH₂. Thiscompound is further identified herein as AGF359.

In a preferred embodiment of this invention, a compound of Formula I,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n= 3, Ar is 2'-fluoro-1,4-phenyl, R is H, and X is CH₂. Thiscompound is further identified herein as AGF347.

In a preferred embodiment of this invention, a compound of Formula I,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n=4, Ar is 2'-fluoro-1,4-phenyl, R is H, and X is CH₂. Thiscompound is further identified herein as AGF355.

In a preferred embodiment of this invention, a compound of Formula I,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n is selected from one of the group of integers consisting of 1,3, and 4, R is H, X is CH₂, and Ar is selected from one of the groupconsisting of (a) 1,4-phenyl, (b) 2'-fluoro-1,4-phenyl, and (c)2,5-thienyl.

In a preferred embodiment of this invention, a compound of Formula I,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n is selected from one of the group of integers consisting of 1,2, 3, and 4, R is CH3, X is CH₂, and Ar is selected from one of thegroup consisting of (a) 1,4-phenyl, (b) 2'-fluoro-1,4-phenyl, and (c)2,5-thienyl.

In a preferred embodiment of this invention, a compound of Formula I,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n is selected from one of the group of integers consisting of 2,3, and 4, R is H, X is selected from one of the group consisting of O,S, NH, NHCHO, NHCOCH₃, and NHCOCF₃ , and Ar is selected from one of thegroup consisting of (a) 1,4-phenyl, (b) 2'-fluoro-1,4-phenyl, and (c)2,5-thienyl.

Another embodiment of this invention provides a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof Formula I, and optionally a pharmaceutically acceptable salt thereof:

Formula I, wherein, R is selected from one of the group consisting of Hand CH₃; n is an integer ranging from 1 to 4; X is selected from one ofthe group consisting of -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and-NHCOCF₃- ; and Ar is selected from one of the group consisting of (a)1,4-phenyl, (b) 2'-fluoro-1,4-phenyl, and (c) 2,5-thienyl. Anotherembodiment of this invention provides the pharmaceutical compositionhaving the compound of Formula I, as described herein, including apharmaceutically acceptable carrier.

In another embodiment of this invention, a compound is provided ofFormula II, and optionally a pharmaceutically acceptable salt thereof:

Formula II, wherein, R is selected from one of the group consisting of Hand CH₃; n is an integer ranging from 1 to 4; X is selected from one ofthe group consisting of -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and-NHCOCF₃-. Those persons skilled in the art will appreciate that any ofthe combination(s) of the moieties identified for the groups of R, andX, and the integers identified for n, are embodiments of the presentinvention for any of the formula(ae) presented for Formula II.

In a preferred embodiment of this invention, a compound of Formula II,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n is 3, R is H, X is CH₂. This compound is identified herein asAGF287.

In a preferred embodiment of this invention, a compound of Formula II,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n is selected from one of the group of integers of 1, 2, and 4,R is H, and X is CH₂.

In a preferred embodiment of this invention, a compound of Formula II,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n is selected from one of the group of integers of 1, 2, 3, and4, R is CH₃, and X is CH₂.

In a preferred embodiment of this invention, a compound of Formula II,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n is selected from one of the group of integers of 1, 2, 3, and4, R is H, and X is selected from one of the group consisting of O, S,NH, NHCHO, NHCOCH₃, and NHCOCF₃.

Another embodiment of this invention provides a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof Formula II, and optionally a pharmaceutically acceptable saltthereof:

Formula II, wherein, R is selected from one of the group consisting of Hand CH₃; n is an integer ranging from 1 to 4; X is selected from one ofthe group consisting of -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and-NHCOCF₃-. A further embodiment of this invention provides apharmaceutical composition having a compound of Formula II, as describedherein, and including a pharmaceutically acceptable carrier.

Another embodiment of this invention provides a compound of Formula III,and optionally a pharmaceutically acceptable salt thereof:

Formula III, wherein, R is selected from one of the group consisting ofH and CH₃; n is an integer ranging from 1 to 4; X is selected from oneof the group consisting of CH and N; Y is selected from one of the groupconsisting of CH and N; and Z is selected from one of the groupconsisting of -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃-.Those persons skilled in the art will appreciate that any of thecombination(s) of the moieties identified for the groups of R, X, Y, andZ, and the integers identified for n, are embodiments of the presentinvention for any of the formula(ae) presented for Formula III.

In a preferred embodiment of this invention, a compound of Formula III,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n is 2, and R is H, and X is either CH or N, Y is either CH orN, and Z is selected from one of the group consisting of CH₂, O, S, NH,NHCHO, NHCOCH₃, and NHCOCF₃. An example of this compound is identifiedherein as AGF315.

In a preferred embodiment of this invention, a compound of Formula III,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n is 3, and R is H, and X is either CH or N, Y is either CH orN, and Z is selected from one of the group consisting of CH₂, O, S, NH,NHCHO, NHCOCH₃, and NHCOCF₃. An example of this compound is identifiedherein as AGF317.

In a preferred embodiment of this invention, a compound of Formula III,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n is an integer selected from the group of 1, 2, 3, and 4, and Ris H, and X is N, Y is CH, and Z is selected from one of the groupconsisting of CH₂, O, S, NH, NHCHO, NHCOCH₃, and NHCOCF₃.

In a preferred embodiment of this invention, a compound of Formula III,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n is an integer selected from the group of 1, 2, 3, and 4, and Ris H, and X is CH, Y is N, and Z is selected from one of the groupconsisting of CH₂, O, S, NH, NHCHO, NHCOCH₃, and NHCOCF₃.

In a preferred embodiment of this invention, a compound of Formula III,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n is an integer selected from the group of 1, 2, 3, and 4, and Ris CH₃, and X is N, Y is CH, and Z is selected from one of the groupconsisting of CH₂, O, S, NH, NHCHO, NHCOCH₃, and NHCOCF₃.

In a preferred embodiment of this invention, a compound of Formula III,and optionally a pharmaceutically acceptable salt thereof, is providedwherein n is an integer selected from the group of 1, 2, 3, and 4, and Ris CH₃, and X is N, Y is CH, and Z is selected from one of the groupconsisting of CH₂, O, S, NH, NHCHO, NHCOCH₃, and NHCOCF₃.

Another embodiment of this invention provides a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof Formula III, and optionally a pharmaceutically acceptable saltthereof:

Formula III, wherein, R is selected from one of the group consisting ofH and CH₃; n is an integer ranging from 1 to 4; X is selected from oneof the group consisting of CH and N; Y is selected from one of the groupconsisting of CH and N; and Z is selected from one of the groupconsisting of -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃-. Afurther embodiment of this invention provides the pharmaceuticalcomposition having the compound of Formula III, as described herein, anda pharmaceutically acceptable carrier.

Another embodiment of this invention provides a method of treating apatient having cancer comprising administering to said patient atherapeutically effective amount of a compound of Formula I, andoptionally a pharmaceutically acceptable salt thereof:

Formula I, wherein, R is selected from the group consisting of H andCH₃; n is an integer ranging from 1 to 4; X is selected from one of thegroup consisting of -CH₂-, O, S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃-; and Ar is selected from one of the group consisting of (a) 1,4-phenyl,(b) 2'-fluoro-1,4-phenyl, and (c) 2,5-thienyl. A preferred embodiment ofthis invention provides a method of treating a patient having cancercomprising administering a therapeutically effective amount of at leastone compound of Formula I selected from the group of AGF 291, AGF 299,AGF300, AGF307, AGF312, AGF318, AGF320, AGF323, AGF331, AGF 347, AGF355, and AGF 359, and optionally a pharmaceutically acceptable saltthereof, to said patient.

Another embodiment of this invention provides a method of treating apatient having cancer comprising administering a therapeuticallyeffective amount of a compound of Formula II, and optionally apharmaceutically acceptable salt thereof:

Formula II, wherein, R is selected from one of the group consisting of Hand CH₃; n is an integer ranging from 1 to 4; and X is selected from oneof the group consisting of -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and-NHCOCF₃-. A preferred embodiment of this invention provides a method oftreating a patient having cancer comprising administering atherapeutically effective amount of a compound of Formula II that isAGF287, and optionally a pharmaceutically acceptable salt thereof, tosaid patient.

Another embodiment of this invention provides a method of treating apatient having cancer comprising administering a therapeuticallyeffective amount of a compound of Formula III, and optionally apharmaceutically acceptable salt thereof:

Formula III, wherein, R is selected from one of the group consisting ofH and CH₃; n is an integer ranging from 1 to 4; X is selected from oneof the group consisting of CH and N; Y is selected from one of the groupconsisting of CH and N; and Z is selected from one of the groupconsisting of -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃-. Apreferred embodiment of this invention provides a method of treating apatient having cancer comprising administering a therapeuticallyeffective amount of at least one compound of Formula III selected fromthe group of AGF 315 and AGF 317, and optionally a pharmaceuticallyacceptable salt thereof, to said patient.

Another embodiment of this invention provides a method of targetingmitochondrial metabolism comprising administering to a cancer patient aneffective amount of at least one compound selected from the group ofFormula I, and optionally a pharmaceutically acceptable salt thereof, ofFormula II, and optionally a pharmaceutically acceptable salt thereof,and of Formula III, and optionally a pharmaceutically acceptable saltthereof:

Formula I, wherein, R is selected from one of the group consisting of Hand CH₃; n is an integer ranging from 1 to 4; X is selected from one ofthe group consisting of -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and-NHCOCF₃- ; and Ar is selected from one of the group consisting of (a)1,4-phenyl, (b) 2'-fluoro-1,4-phenyl, and (c) 2,5-thienyl; and

Formula II, wherein, R is selected from one of the group consisting of Hand CH₃; n is an integer ranging from 1 to 4; and X is selected from oneof the group consisting of -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and-NHCOCF₃-; and

Formula III, wherein R is selected from one of the group consisting of Hand CH₃; n is an integer ranging from 1 to 4; X is selected from one ofthe group of CH and N; Y is selected from one of the group of CH and N;and Z is selected from one of the group consisting of -CH₂- , O, S,-NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃-.

Another embodiment of this invention provides a method of targetingSHMT2 and MTHFD2 comprising administering to a cancer patient aneffective amount of at least one compound selected from the group ofFormula I, and optionally a pharmaceutically acceptable salt thereof, ofFormula II, and optionally a pharmaceutically acceptable salt thereof,and of Formula III, and optionally a pharmaceutically acceptable saltthereof:

Formula I, wherein, R is selected from one of the group consisting of Hand CH₃; n is an integer ranging from 1 to 4; X is selected from one ofthe group consisting of -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and-NHCOCF₃- ; and Ar is selected from one of the group consisting of (a)1,4-phenyl, (b) 2'-fluoro-1,4-phenyl, and (c) 2,5-thienyl; and

Formula II, wherein, R is selected from one of the group consisting of Hand CH₃; n is an integer ranging from 1 to 4; and X is selected from oneof the group consisting of -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and-NHCOCF₃-; and

Formula III, wherein R is selected from the group consisting of H andCH₃; n is an integer ranging from 1 to 4; X is selected from one of thegroup consisting of CH and N; Y is selected from one of the groupconsisting of CH and N; and Z is selected from one of the groupconsisting of -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃-.

Those skilled in the art shall understand that chemical structures ofFormulae I, II, and III, are preferred examples of the compounds of thisinvention and that tautomers of Formulae I, II, and III are alsoembodiments of compounds of Formula I, Formula II, and Formula III,respectively. Those skilled in the art understand that chemicalstructures are often drawn as one tautomeric form over another. Thisinvention provides for several tautomeric forms of the oxygen attachedat the fourth carbon of the pyrimidine six membered ring of thecompounds of this invention. The tautomeric forms (i.e. oxygen withdouble bond, or -OH) provide additional structural embodiments that willbe appreciated by those skilled in the art.

In certain embodiments of the invention, the novel compounds includeFormula I, Formula II, and Formula III, as described herein, and includepharmaceutically acceptable salts of these compounds, and include forexample but not limited to, hydrochloride chloride (HCl) salts (or otheracids) of these compounds.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the patients being treated, each unitcontaining a predetermined quantity or effective amount of a compound ofthe present invention to produce the desired effect in association witha pharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the particularcompound and the particular effect, or therapeutic response, that isdesired to be achieved.

Compounds of Formula I, II, or III, or pharmaceutically acceptablesalts, or hydrates thereof, can be administered to a patient (an animalor human) via various routes including parenterally, orally orintraperitoneally. Parenteral administration includes the followingroutes that are outside the alimentary canal (digestive tract):intravenous; intramuscular; interstitial, intraarterial; subcutaneous;intraocular; intracranial; intraventricular; intrasynovial;transepithelial, including transdermal, pulmonary via inhalation,ophthalmic, sublingual and buccal; topical, including dermal, ocular,rectal, or nasal inhalation via insufflation or nebulization. Specificmodes of administration shall depend on the indication. The selection ofthe specific route of administration and the dose regimen is to beadjusted or titrated by the clinician according to methods known to theclinician in order to obtain the optimal clinical response. The amountof compound to be administered is that amount which is therapeuticallyeffective. The dosage to be administered to a patient shall depend onthe characteristics of the patient being treated, including for example,but not limited to, the patient’s age, weight, health, and types andfrequency of concurrent treatment, if any, of any other chemotherapeuticagent(s), all of which is determined by the clinician as one skilled inthe art.

Compounds of Formula I, II, or III, or a pharmaceutically acceptablesalt, or hydrate thereof, that are orally administered can be enclosedin hard or soft shell gelatin capsules, or compressed into tablets.Compounds also can be incorporated with an excipient and used in theform of ingestible tablets, buccal tablets, troches, capsules, sachets,lozenges, elixirs, suspensions, syrups, wafers and the like. Compoundsof Formula I, II, or III can be in the form of a powder or granule, asolution or suspension in an aqueous liquid or non-aqueous liquid, or inan oil-in-water emulsion.

The tablets, troches, pills, capsules and the like also can contain, forexample, a binder, such as gum tragacanth, acacia, corn starch; gelatingexcipients, such as dicalcium phosphate; a disintegrating agent, such ascorn starch, potato starch, alginic acid and the like; a lubricant, suchas magnesium stearate; a sweetening agent, such as sucrose, lactose orsaccharin; or a flavoring agent. When the dosage unit form is a capsule,it can contain, in addition to the materials described above, a liquidcarrier. Various other materials can be present as coatings or tootherwise modify the physical form of the dosage unit. For example,tablets, pills, or capsules can be coated with shellac, sugar or both. Asyrup or elixir can contain the active compound, and sucrose as asweetening agent, methyl and propylparabens as preservatives, a dye andflavoring, for example. Any material used in preparing any dosage unitform should be pharmaceutically pure and substantially non-toxic.Additionally, the compounds of Formulae I, II, and III, or apharmaceutically acceptable salt, or hydrate of the compounds ofFormulae I, II, and III, can be incorporated into sustained-releasepreparations and formulations.

The compounds of Formula I, Formula II, or Formula III, or apharmaceutically acceptable salt, or hydrate thereof, can beadministered to the central nervous system, parenterally orintraperitoneally. Solutions of the compound as a free base or apharmaceutically acceptable salt can be prepared in water mixed with asuitable surfactant, such as for example, but not limited to,hydroxypropylcellulose. Dispersions also can be prepared in glycerol,liquid polyethylene glycols and mixtures thereof, and in oils. Underordinary conditions of storage and use, these preparations can contain apreservative and/or antioxidants to prevent the growth of microorganismsor chemical degeneration.

The pharmaceutical forms suitable for injectable use include, withoutlimitation, sterile aqueous solutions or dispersions and sterile powdersfor the extemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It can be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

Compounds of the present invention may be contained within, mixed with,or associated with, a suitable (acceptable) pharmaceutical carrier foradministration to a patient according to the particular route ofadministration desired. Suitable or acceptable pharmaceutical carriersrefer to any pharmaceutical carrier that will solubilize the compoundsof the present invention and that will not give rise to incompatabilityproblems, and includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic agents, absorptiondelaying agents, and the like. The use of such suitable or acceptablepharmaceutical carriers is well known by those skilled in the art.Preferred carriers include sterile water, physiologic saline, and fivepercent dextrose in water. Examples of other suitable or acceptablepharmaceutical carriers include, but are not limited to, ethanol, polyol(such as propylene glycol and liquid polyethylene glycol), suitablemixtures thereof, or vegetable oils. The proper fluidity can bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size (in the case of adispersion) and by the use of surfactants. The prevention of the actionof microorganisms can be brought about by various antibacterial andanti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.

Sterile injectable solutions are prepared by incorporating a compound ofFormula I, II, or III, in the required amount in the appropriate solventwith various of the other ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the sterilized compound of Formula I, II, or III, intoa sterile vehicle that contains the basic dispersion medium and any ofthe other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze drying.

Pharmaceutical compositions which are suitable for administration to thenose and buccal cavity include, without limitation, self-propelling andspray formulations, such as aerosol, atomizers and nebulizers.

The therapeutic compounds of Formula I, II, or III, as described herein,can be administered to a patient alone or in combination withpharmaceutically acceptable carriers or as pharmaceutically acceptablesalts, or hydrates thereof, the proportion of which is determined by thesolubility and chemical nature of the compound, chosen route ofadministration to the patient and standard pharmaceutical practice.

The present invention is more particularly described in the followingnon-limiting examples, which are intended to be illustrative only, asnumerous modifications and variations therein will be apparent to thoseskilled in the art.

EXAMPLES

Animal studies indicate in-vivo potent antitumor activity againstpancreatic cancer in mice for compound of the present inventionidentified by AGF291. The preliminary results provided herein includepancreatic cancer data for AGF287 and AGF291. FIG. 6 shows data forgrowth inhibitions by 5- and 6-substituted pyrrolo[2,3-d]- andpyrrolo[3,2-d]pyrimidine compounds of this invention, compared togemcitabine (GEM) toward pancreatic cancer cell lines expressing themajor facilitative folate transporters. Additional results and data areshown in FIGS. 5, 6, 8, and 11 .

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification beconsidered as exemplary only. Furthermore, the examples are meant to beillustrative of certain embodiments of the invention and are notintended to be limiting as to the scope of the invention.

Genes encoding one-carbon (C1) metabolism enzymes in the mitochondriaand cytosol are consistently upregulated across multiple cancer types.Whereas multi-targeted cytosolic C1 metabolism inhibitors such aspemetrexed are used clinically, there are no current anti-cancer drugsthat target upstream mitochondrial C1 metabolism. The present inventionprovides novel, multi-targeted small molecule inhibitors ofmitochondrial C1 metabolism at serine hydroxymethyltransferase (SHMT) 2,and cytosolic C1-dependent purine biosynthesis(5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase andglycinamide ribonucleotide formyltransferase) and SHMT1 asbroad-spectrum antitumor agents toward lung, colon and pancreatictumors. In vivo anti-tumor efficacy is demonstrated in a pancreas cancermodel. The compounds and pharmaceutically acceptable salts thereof aredual-targeting mitochondrial and cytosolic C1 metabolism for cancer. Thecompounds and pharmaceutically acceptable salts there of the presentinvention overcome resistance to current anticancer therapies.

Folate-dependent one-carbon (C1) metabolism is compartmentalized in themitochondria and cytosol and supports cell growth through nucleotide andamino acid biosynthesis. Mitochondrial C1 metabolism including serinehydroxymethyltransferase (SHMT) 2 provides glycine, NAD(P)H, and C1units for cytosolic biosynthetic reactions, and is implicated in theoncogenic phenotype across a wide range of cancers. Whereasmulti-targeted inhibitors of cytosolic C1 metabolism such as pemetrexedare used clinically, there are currently no anticancer drugs thatspecifically target upstream mitochondrial C1 metabolism. We usedmolecular modeling to design novel small-moleculepyrrolo[3,2-d]pyrimidine inhibitors targeting mitochondrial C1metabolism at SHMT2 as potential broad-spectrum antitumor agents. Invitro antitumor efficacy was established with the lead compounds of thepresent invention, namely, AGF291, AGF320, AGF347, toward lung, colon,and pancreatic cancer models. Intracellular targets were identified byglycine/nucleoside protection and targeted metabolomics with an isotopetracer, with confirmation by in vitro assays with purified enzymes. Inaddition to targeting SHMT2, inhibition of the purine biosyntheticenzymes, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferaseand/or glycinamide ribonucleotide formyltransferase, and SHMT1 in thecytosol was established. The compounds and pharmaceutically acceptablesalts thereof of the present invention have first-in-class in vivoantitumor efficacy with compound AGF291 toward the MIA PaCa-2 pancreaticadenocarcinoma xenograft model in severe-combined immunodeficient mice,providing compelling proof-of-concept of the therapeutic potential ofmulti-targeting SHMT2 and cytosolic C1 enzymes by the series ofcompounds of the present invention. Our results providestructure-activity relationships and identify novel drug compounds forfurther development as multi-targeted anti-tumor agents with impressivepotential to overcome resistance to current therapies.

The present invention provides a novel series of 5-substitutedpyrrolo[3,2-d]pyrimidine antifolate analogs with primary inhibition ofSHMT2, and secondary inhibition of AICARFTase and/or GARFTase, as wellas of SHMT1. Direct inhibition of de novo purine biosynthesis downstreamof SHMT2 is potentiated by loss of mitochondrial C1 metabolism,resulting from primary SHMT2 inhibition, reflecting the impact oflimiting glycine and 10-formyl-THF. The present invention provides novelcompounds, such as for example, AGF291, AGF320, and AGF347, withbroad-spectrum in vitro anti-tumor efficacies including H460 non-smallcell lung cancer (NSCLC), HCT116 colon cancer, and MIA PaCa-2 pancreaticcancer (PaC) cells. For AGF291, in vitro findings were extended in vivoto MIA PaCa-2 tumor xenografts in severe compromised immunodeficient(SCID) mice, providing compelling proof-of-concept of the therapeuticpotential of multi-targeted SHMT2 therapeutics for cancer.

Table 1 shows the results of in vitro assays, N-terminally His-taggedproteins were purified including GARFTase (transformylase domain;residues 100-302), ATIC (AICARFTase/IMP cyclohydrolase), SHMT2 andMTHFD2. For SHMT2, a coupled SHMT2-MTHFD2 assay was used with an NADHreadout. There was no inhibition of MTHFD2 by the analogs. AGF291 andAGF320 (as monoglutamates) inhibited SHMT2 (K_(i)s of 0.82 and 0.28 µM,respectively). (AGF347 and AGF359,

TABLE 1 K_(I) values (µM) for in vitro inhibition of the indicatedfolate metabolic enzymes by pyrrolopyrimidine compounds. Analog K_(i)(µM) SHMT2 GARFTase AICARFTase MTHFD2 AGF291 0.82 ± 0.49 Not detected10.91 ± 6.07 Not detected AGF320 0.28* 0.33 ± 0.22 6.96 ± 4.98 Notdetected AGF347 ND 3.13 ± 0.66 3.72 ± 1.61 Not detected AGF94 ND 0.47 ±0.11 Not detected ND “Not detected” means no inhibition was detected upto 100 µM. “ND” means not determined. * designates one experiment.

testing ongoing). We also assayed inhibition of GARFTase and AICARFTaseby AGF291, AGF320, and AGF347 (76, 77). For GARFTase and AICARFTase,inhibition by AGF291, AGF320 and AGF347 paralleled that measured bymetabolomics assays. These results confirm that SHMT2 and the purinebiosynthetic enzymes GARFTase and AICARFTase are direct targets of ourlead pyrrolo[3,2-d]pyrimidine compounds.

Table 2 provides proliferation results for engineered CHO and humantumor cells. Proliferation assays were performed with engineered Chinesehamster ovary (CHO) cell lines R2 (transporter-null), PC43-10 (expressesRFC only), RT16 (FRα only), and R2/PCFT4 (PCFT only) cultured infolate-/glycine-free RPMI1640 supplemented with 25 nM leucovorin.Additional results are shown for HCT116 (colon tumor cells (CTC)),IGROV1 (epithelial ovarian cancer (EOC) cells), H460 (non-small celllung cancer (NSCLC) cells), MIAPaCa-2 (pancreatic cancer (PaC) cells),and HPAC (PaC). Results are shown as mean IC50s (± standard deviation;n=4).

TABLE 2 Proliferation results for engineered CHO and human tumor cellsCompound Series FIG. 2 n R IC50s (SE) (nMolar) R2 (null) (CHO) PC43-10(RFC) (CHO RT16 (FR alpha) (CHO) R2/PCFT4 (CHO) HCT116 (CTC) IGROV1(EOC) H460 (NSCLC) MIA PaCa-2 (PaC) HPAC (PaC) AGF291 Benzoyl 3 H >1000454(87) 117(10) 282(23) 2266 (450) 443 (88) 461 (163) 3664 (721) 301(22) AGF320 Thienoyl 5 NA >1000 >1000 156(18) 694(56) 737 (195) 401(140) 573 (145) 2266 (400) 190 (21) AGF347 Benzoyl 4 F >1000 224(17)9.4(0.1) 479(64) 437 (180) 62 (43) 214 (88) 1381 (182) 764 (73) AGF359Benzoyl 3 F >1000 233(25) 525 (229) 98 (17) ND 229 (37) ND >1000 167.45(20.45) Abbreviations: CHO, Chinese hamster ovary; EOC, epithelialovarian cancer; NSCLC, non-small cell lung cancer; PaC, pancreaticcancer

Design of 5-substituted pyrrolo[3,2-d]pyrimidine antifolates of thepresent invention targeting mitochondrial C1 metabolism at SHMT2 isprovided herein. Given the association of mitochondrial C1 metabolismwith malignancy (14-17) , including reports of SHMT2 as a potential“onco-driver” (15, 22), it was of interest to develop inhibitors ofSHMT2 as potential antitumor agents. We initially looked for potentialSHMT2 inhibitors among the known arsenal of classic antifolates(including methotrexate, pemetrexed, raltitrexed and lometrexol) (6-8)and previous GARFTase and AICARFTase inhibitors from published studies(23-29). We tested these compounds for inhibition of proliferation ofChinese hamster ovary (CHO) cells engineered from a folatetransporter-null (MTXRIIOua^(R)2-4) CHO subline (hereafter, R2) (30) toexpress human PCFT (R2/PCFT4) (24), as we reasoned that tumor-selectiveuptake of a putative SHMT2 inhibitor by PCFT (31) would be desirable.Since inhibition of mitochondrial C1 metabolism (at SHMT2) would beexpected to induce glycine auxotrophy, as seen in SHMT2 KO (KO) cells(20), and mitochondrial C1 metabolism generates C1 units (i.e., formate)critical for downstream nucleotide biosynthesis, these experiments wereperformed in glycine- and nucleoside-free media, and the protectiveeffects of added glycine (130 µM), adenosine (60 µM), and/or thymidine(10 µM) were determined. All these compounds inhibited cellproliferation and nucleotide biosynthesis (reflected in adenosine and/orthymidine protection), however, glycine was not protective with orwithout nucleoside additions. Thus, the known compounds of thebackground art were not useful. We are aware of the structural featuresof our previous 5-substituted pyrrolo[2,3-d]pyrimidine benzoyl andthienoyl compounds (28, 32) and with those of 5,10-methylene-THF (SHMT2product) and 5-formyl-THF (SHMT inhibitor) (33), see FIG. 2 . Thepresent invention provides 5-substituted pyrrolo[3,2-d]pyrimidinecompounds and includes 3-5 bridge carbons linked to benzoyl, namelycompounds of the present invention identified herein as AGF291, AGF300,and AGF299, or thienoyl, namely the compounds of the present inventionidentified herein as AGF331, AGF318, and AGF320. Further, the presentinvention provides compounds having a 2' fluorine substitution forincreasing the inhibitory potencies of pyrrolo[2,3-d]pyrimidinecompounds, such as for example, the 2'-fluorinated compounds of thepresent invention identified as AGF300 and AGF299, as well as AGF347 andAGF355. All compounds, described, were docked into human SHMT2 (seeFIGS. 12A-12D) and docking scores (see Table S1) were better than forthe previously reported pyrazolopyran inhibitor SHIN1 (21) (-5.58kcal/mol) upon re-docking.

TABLE S1 Docking scores of antifolates for intracellular drug targets(kcal/mol) Compound SHMT2 SHMT1 AGF291 -7.69 -10.45 AGF300 -7.58 -7.17AGF299 -7.42 -7.48 AGF347 -10.06 -8.90 AGF355 -6.55 -6.45 AGF331 -6.93-7.19 AGF318 -6.43 -7.03 AGF320 -8.32 -11.14 Molecular modeling wasperformed for all analogs with the structure of human SHMT2 (PDB: 5V7l)(1) and for rabbit SHMT1 (PDB: 1LS3) (2), using the induced fit dockingprotocol of Maestro (44, 45).

Discovery of 5-substituted pyrrolo[3,2-d]pyrimidine antifolates of thepresent invention which target mitochondrial C1 metabolism. Wesynthesized the novel compounds (see below synthesis schemes) andscreened these compounds for inhibition of cell proliferation. Weinitially assessed inhibition by these compounds (from 0 to 1000 nM)toward PCFT-expressing R2/PCFT4 CHO cells and an isogenic CHO sublineengineered to express human RFC (PC43-10) (23). Results were compared tothose for folate transporter-null R2 CHO cells as a negative control.IC₅₀ values for “active” compounds are shown in FIG. 6 for each of thecompounds of the present invention, along with those for AGF94 (a “pure”GARFTase inhibitor) (27). Active compounds of the present inventiontoward R2/PCFT4 and PC43-10 cells included AGF291, AGF320, AGF331 andAGF347, with AGF291 showing selectivity (~1.6-fold) toward PCFT overRFC. These compounds were further tested with human tumor cell lines,including H460 NSCLC, HCT116 colon cancer, and MIA PaCa-2 PaC cells,characterized by expression of PCFT and RFC, but not FRα (34, 35) (seeFIG. 13 ). IC₅₀ values for growth inhibition are in FIG. 6 . Althoughthere were notable differences in drug sensitivities among the assortedtumor models, the compounds identified by AGF291, AGF320 and AGF347 wereconsistently the most active.

We again used glycine/nucleoside protection studies (above) in H460,HCT116, IGROV1 and MIA PaCa-2 cells treated with AGF291, AGF320, orAGF347 to identify the targeted pathways. The results were compared tothose for AGF94 and are shown in FIG. 3 for H460 cells. Adenosine (60µM) was fully protective up to 10 µM AGF94, whereas glycine (130 µM) hadno effect. We also tested the protective effects of AICA (320 µM) whichis metabolized to AICAR (ZMP) (AICARFTase substrate), thus circumventingthe GARFTase step in de novo purine biosynthesis (27) (see FIG. 1 ). AsAICA was completely protective, GARFTase must be the intracellulartarget for AGF94 (27). For AGF291, AGF347, and AGF320, neither glycinenor adenosine alone was fully protective. However, combined adenosineand glycine was substantially protective for all compounds (see FIG. 3). These results strongly suggest that these compounds of the presentinvention target both mitochondrial C1 metabolism and cytosolic de novopurine biosynthesis. Thymidine provided no protection from any of thecompounds and did not increase the extent of protection by glycine andadenosine. For some of the compounds, notably AGF320, growth inhibitionwas modestly (and incompletely) reversed by AICA (with glycine) (seeFIG. 3 ), suggesting a secondary intracellular target, most likelyGARFTase. Analogous results were obtained with HCT116 and MIA PaCa-2tumor cells (see FIG. 14 ).

Collectively, these results establish that the compounds of the presentinvention, namely, AGF291, AGF320, and AGF347 target both mitochondrialand cytosolic C1 metabolism.

Identification of the mitochondrial enzyme target for AGF291, AGF320,and AGF347 by targeted metabolomics. To further confirm theintracellular enzyme targets of the lead compounds of this invention,namely, AGF291, AGF320, and AGF347, we performed targeted metabolomicswith liquid chromatography-mass spectrometry (LC-MS) and[2,3,3-²H]serine tracer in HCT116, H460 and MIA PaCa-2 tumor cells. Thecells were processed for LC-MS analyses of total serine, and of M+3,M+2, M+1, and M+0 (unlabeled) serine (where M+n represents species withn deuterium atoms). The results with the drug-treated cells werecompared to those for untreated cells. For WT HCT116 cells, the resultswere compared to those for SHMT2 KO, MTHFD2 KO, and SHMT1 KO cells (KOof SHMT2, MTHFD2, and SHMT1, respectively (20)). For H460 cells,controls included non-targeted control (NTC) and SHMT2 shRNA knockdown(KD) cells.

Total serine pools were elevated ~10-fold in the HCT116 sublines withmitochondrial C1 KOs (SHMT2 KO and MTHFD2 KO) relative to WT controls(see FIG. 4B). KO of cytosolic SHMT1 (SHMT1 KO) had no impact on totalserine pools. This establishes that SHMT2 rather than SHMT1 is theprimary catabolic enzyme for serine in the tumor cells (20). In AGF291-,AGF320- and AGF347-treated HCT-116 cells, total serine also increased~10-fold, indicating a profound loss of serine catabolism. Analogousresults were seen with the H460 and MIA PaCa-2 cell lines, including theH460 SHMT2 KD cells (see FIGS. 15A and 15B).

The flux of [²H]metabolites originating from [2,3,3-²H]serine tracer isdepicted in FIG. 4A. In tumor cells, C1 metabolism flows in a clockwisemanner, with serine catabolized in mitochondria (starting with SHMT2)and regenerated in the cytosol (via SHMT1) (20). The reactions arereversible (e.g., serine can be resynthesized from formate in themitochondria (20)). The compartmental metabolism of serine can berevealed by analyzing the isotope tracing (“scrambling”) patterns from[2,3,3-²H]serine in HCT116 cells (20, 21), including unmetabolized (i.e.M+3), and M+2 and M+1 serine resulting from 5,10-methylene-THF (M+2) andformate/10-formyl-THF (M+1), respectively (see FIG. 4A).

In WT HCT116 cells treated with [2,3,3-²H]serine, most of the serine wascatabolized (loss of M+3 serine was ~90%) (see FIG. 4C) and the labelthat remained was mostly incompletely labeled (scrambled) serine,reflecting resynthesized serine from labeled glycine (M+1 fraction; ~30%of total serine) (20). SHMT1 KO led to a drop (~60%) in the M+1 fractionwithout altering the M+2 serine fraction, consistent with SHMT1 in thecytosol being responsible for bulk synthesis of serine from glycine whenSHMT2 is active (20). In SHMT2 KO cells, the M+3 fraction was >60% ofthe total serine (see FIG. 4C, consistent with a profound loss of serinecatabolism. Reflecting this and the depletion of formate downstream ofSHMT2, there was ~90% decreased M+1 serine (20). Further, the M+2 serinefraction also decreased (~25%) compared to WT cells, reflecting depleted5,10-methylene-THF. Although similar results were seen for the M+1 andM+3 serine fractions with MTHFD2 KO cells (decreased ~75% and increased3.5-fold, respectively, from WT levels), M+2 serine was increased(~1.7-fold) compared to that resulting from SHMT2 KO (see FIG. 4C). Thisreflects accumulation of 5,10-methylene-THF when MTHFD2 is lost (i.e.,the M+2 serine isotopomer is generated through reversible activity ofSHMT2 via proton abstraction). Thus, changes in serine isotope labelingfrom [2,3,3-²H]serine are diagnostic for the specific perturbations infolate metabolism, and also inform upon the particular enzymatic stepthat is inhibited.

Treatment of WT HCT116 cells with compounds of the present invention,namely, AGF291, AGF320 or AGF347 closely recapitulated the effects ofthe SHMT2 KO, including a substantial M+3 serine fraction (~55-60% oftotal serine) and decreased M+2 serine (~2-3-fold) compared to WT cells,accompanied by nearly complete loss of M+1 serine (see FIG. 4E).Analogous results were obtained with SHMT2 KD and drug-treated WT H460cells, and with MIA PaCa-2 cells treated with AGF291 (see FIG. 15C andFIG. 15D, respectively). Importantly, these results identify SHMT2 asthe mitochondrial target for compounds of the present invention AGF291,AGF320 and AGF347.

Identification of cytosolic targets for compounds of the presentinvention AGF291, AGF320, and AGF347 by targeted metabolomics. BothGARFTase and AICARFTase require 10-formyl-THF derived from formate, mostof which is generated via mitochondrial C1 metabolism from serine (20)(see FIGS. 1 and 4A). Consistent with this, loss of SHMT2 in H460 SHMT2KD cells induced significant increases in purine intermediates which aredependent on C1 pools (i.e., 10-formyl-THF), including GAR (GARFTasesubstrate; 21-fold) (see FIG. 4D) and AICAR (AICARFTase substrate;65-fold) (see FIG. 4E). Likewise, treatment with AGF291, AGF320, andAGF347 (10 µM) all increased GAR (10-2300-fold) and AICAR (40-1500-fold)relative to untreated controls (see FIGS. 4D and 4E). Similar increasesin GAR and AICAR pools resulted in drug-treated HCT116 and MIA PaCa-2cells (see FIGS. 15E, 15F, 15I, and 15J). For the HCT116 sublines, theincreases in GAR and AICAR upon drug treatments generally exceeded thoseresulting from the SHMT2 KO.

To assess the possibility that the compounds of the present invention,namely, AGF291, AGF320 and AGF347 directly inhibit cytosolic enzymetargets in de novo purine biosynthesis (i.e., GARFTase and/orAICARFTase), we treated the H460 cells with 1 mM formate, to replenishthe cytosolic C1 pool while circumventing the mitochondrial C1 pathway.We reasoned that formate treatment of the SHMT2 KD cell line shouldrestore levels of GAR and AICAR to those seen in NTC (WT) cells.However, if the cytosolic enzymes were directly inhibited, formateshould not effectively reverse accumulation of GAR and/or AICAR. Indeed,in H460 SHMT2 KD cells, treatment with formate completely reversedelevated GAR (see FIG. 4D) and AICAR (see FIG. 4E) accumulations to NTClevels. However, for drug-treated H460 cells, reversal by formate wasincomplete, albeit to different extents for different compounds. WithGAR, the extent of formate reversal was in the order, AGF291 > AGF347 >AGF320, whereas for AICAR, the rank order was AGF347 > AGF320 > AGF291.These results strongly implicate direct targeting of GARFTase and/orAICARFTase by these novel inhibitors in addition to SHMT2.

Targeted Metabolomics: Identification of SHMT1 as a target forpyrrolopyrimidine compounds of this invention. As SHMT2 (UniProtKB:P34897) maintains 66% sequence identity (36) to SHMT1 (UniProtKB:34896), we considered the possibility that compounds of the presentinvention AGF291, AGF320 and AGF347 may also target SHMT1 in thecytosol, although this would be secondary to inhibition of SHMT2. Bymolecular modeling (see FIGS. 12F-12H), these compounds bound to rabbitSHMT1 (UniProtKB: 07511, 93% sequence homology with human SHMT1(37))with docking scores from -8.9 to -11.14 kcal/mol (see Table S1).

To gauge potential SHMT1 inhibition by our compounds, we traced[2,3,3-²H]serine into dTMP (dTTP), via C1 transfer from5,10-methylene-THF to dUMP by TS (20) (see FIG. 4A). WT H460 cellsincubated with [2,3,3-²H]serine generated M+1 dTTP without M+2 dTTP (seeFIG. 4G), confirming [2,3,3-²H]serine metabolism through themitochondrial C1 pathway to [²H]formate into TTP (M+1) (20). Knockdownof SHMT2 (SHMT2 KD) induced a robust M+2 dTTP signal, reflecting thereverse-flux (serine → glycine) through SHMT1 in the cytosol (20). Inspite of compelling evidence for direct SHMT2 targeting (above),treatment with AGF291, AGF320, or AGF347 (10 µM) resulted in M+1 dTTPwithout M+2 dTTP (see FIG. 4G). This was accompanied by reduced (60-70%)dTTP pools (see FIG. 4F). Analogous results were obtained with both theHCT116 and MIA PaCa-2 sublines (see FIGS. 15G, 15H, 15K, and 15L).Treatment with 1 mM formate to elevate cytosolic C1 pools abolished ²Hincorporation from [2,3,3-²H]serine into dTTP (see FIG. 4G) for thedrug-treated cells, this was accompanied by only partial restoration ofdTTP (see FIG. 4F). For the SHMT2 KD cells, loss of SHMT2 resulted insuppressed dTTP (likely due to a decreased cytosolic C1 pool), and adTTP “overshoot” when excess formate was provided.

As adenosine rather than thymidine (combined with glycine) wasprotective from growth inhibition by the compounds of the presentinvention, see FIGS. 3 and 14 , the reduced dTTP pool (see FIG. 4F),combined with the absence of M+2 dTTP from [2,3,3-²H]serine (see FIG.4G), is most consistent with the direct targeting of SHMT1, in additionto SHMT2. Although our results are explained by targeting of both SHMT1and SHMT2, the relative magnitudes of these inhibitions are difficult toassess by metabolomics assays alone.

Enzymology: To confirm the multiple enzyme targets (SHMT2, GARFTase,AICARFTase, and SHMT1) identified from our metabolomics experiments, weperformed in vitro assays using purified recombinant enzymes. N-terminalHis-tagged proteins were purified including GARFTase (formyltransferasedomain; residues 100-302), ATIC (AICARFTase/IMP cyclohydrolase), SHMT2,SHMT1, and MTHFD2. GARFTase and AICARFTase assays were carried out aspreviously reported (38, 39) with slight modifications, whereas SHMT1/2and MTHFD2 assays were developed for this study. For MTHFD2, using NADHas a readout, none of our lead molecules were inhibitory. For SHMT1 andSHMT2, a coupled enzyme assay with MTHFD2 was used. Our resultsdemonstrate primary targeting of SHMT2 by “monoglutamyl” AGF291 andAGF320 (K_(I)s of 0.89 µM and 0.28 µM, respectively (Table 1). Therelative K_(i)s for GARFTase and AICARFTase corroborate our metabolomicsresults (see FIGS. 4D and 4E), for which AGF320 was the most potentGARFTase inhibitor and AGF291 was the most potent AICARFTase inhibitor.These results confirm that SHMT2, and the purine biosynthetic enzymesGARFTase, and AICARFTase, are direct targets of our leadpyrrolo[3,2-d]pyrimidine compounds. As a control, AGF94 was tested andfound to inhibit only GARFTase, with no inhibition for AICARFTase,SHMT2, or MTHFD2.

In vivo anti-tumor efficacy study with MIA PaCa-2 tumor xenografts.Based on in vitro efficacies with AGF291 toward various tumor cells (seeFIG. 6 ), we performed an in vivo efficacy trial in comparison with thegemcitabine (GEM). Both AGF291 and GEM were efficacious, with mediantumor burdens on day 14 of 256 mg (range 75-851 mg) and 255 mg (range63-322 mg), respectively, compared to 1321 mg (range 685-1465 mg) forthe control cohort. T/C values were 19% for AGF291 and 26% for GEM (seeFIG. 5 ). Tumor growth delays (median T-C to reach 1000 mg) of 10.5 daysfor AGF291 and 7.5 days for GEM were recorded. AGF291 and GEM were welltolerated with modest weight losses (9% median nadir on day 17 and 12%median nadir on day 6, respectively) that were completely reversibleafter cessation of therapy. Thus, at equitoxic dose levels, AGF291showed better anti-tumor efficacy than GEM, with a 20-fold decreaseddose requirement and no acute or long-term toxicities other thanreversible weight loss.

Table S2 shows in vivo efficacies of AGF291 and gemcitabine toward theMIA PaCa-2 xenografts. Female ICR SCID mice (10 weeks old; 19 g averagebody weight) were implanted bilaterally with human MIA PaCa-2 tumors.For the efficacy trial, the mice were maintained on either afolate-deficient diet from Harlan-Teklad (TD.00434) starting 14 daysbefore subcutaneous tumor implant to ensure serum folate levels wouldapproximate those of humans. Beginning on day 3 following subcutaneousimplantation, the mice were dosed as follows: compound of this inventionAGF291, every 6 days for three injections (Q6dx3) at 7.75 mg/kg/inj.(injection), total dose 23.5 mg/kg; and gemcitabine, every 4 days forthree injections (Q4dx3) at 120 mg/kg/inj., total dose 480 mg/kg). Themice were weighed and tumors were measured with a caliper two-to-threetimes weekly; mice were sacrificed when the cumulative tumor burdenreached 1500 mg. Tumor weights were estimated from two-dimensionalmeasurements, where tumor mass (in mg) = (a x b²)/2, and a and b are thetumor length and width in mm, respectively. The tumor masses from bothtumors on each mouse were added together, and the total mass per mousewas used for calculations of anti-tumor activity. Quantitativeend-points include: (i) tumor growth delay [T-C, where T is the mediantime in days required for the treatment group tumors to reach apredetermined size (e.g., 1000 mg), and C is the median time in days forthe control group tumors to reach the same size; tumor-free survivorsare excluded from these calculations]; and (ii) T/C (in percent) whentreatment (T) and control (C) groups for the control groups reached 700mg in size (exponential growth phase). The median value of each groupwas determined (including zeros). Mouse weights were monitored as agauge of drug toxicity.

TABLE S2 Antitumor Efficacy Evaluation of AGF-291 and GemcitabineAgainst Early Stage Human MIA PaCa-2 Pancreatic Adenocarcinomaxenografts in Female NCR SCID Mice Treatment Drug Route Schedule TotalDosage mg/kg Mean Body Weight Loss (g/mouse) Percent Body Weight LossMedian Tumor Burden in mg on d16 (range) T/C% Tumor Free on d52 Time to1000 mg in days (range) Tumor Growth Delay (T-C) (days) No treatment-0.2 -1.0 1726 (1213-2038) 0/5 12.5 (12.5-15.0) AGF291 IV Q6dx3 Start d323.5 -1.8 -8.9 334 (0-1221) 19 0/5 23.0 (15.0-51) 10.5 Gemcitabine IVQ4dx4 Start d3 480 -2.4 -12.2 445 (150-527) 26 0/5 20 (19-21) 7.5

Most recently, we performed an in vivo study of MIA PaCa-2 xenograftswith AGF347 (FIG. 6 ). In vivo efficacies of AGF347 and GEM toward theMIA PaCa-2 PaC xenografts. Female ICR SCID mice (10 weeks old; 19 gaverage body weight) were implanted bilaterally with human MIA PaCa-2PaC tumors. Beginning on day 3 following subcutaneous implantation, themice were dosed as follows: AGF347, Q2dx8 at 15 mg/kg/inj, total dose120 mg/kg; and GEM, Q4dx4 at 120 mg/kg/inj, total dose 480 mg/kg). T/Cvalues were 19% for AGF291 and 26% for GEM. For AGF347, the T-C (1000mg) was 54 days and 1/5 mice was disease-free at 122 days.

Methods

Chemicals. [¹⁴C]Formate (50-60 mCi/mmol) was purchased from MoravekBiochemicals (Brea, CA). [2,3,3-²H, 98%]L-Serine was purchased fromCambridge Isotope Laboratories, Inc. (Andover, MA). Leucovorin [(6R,S)5-formyl tetrahydrofolate (THF)] was provided by the Drug DevelopmentBranch, National Cancer Institute (Bethesda, MD). Pemetrexed (Alimta)(PMX) was purchased from LC Laboratories (Woburn, MA). Lometrexol(5,10-dideaza-5,6,7,8-tetrahydrofolate) was obtained from Eli Lilly andCo. (Indianapolis, IN). Raltitrexed was obtained from AstraZenecaPharmaceuticals (Maccesfield, Cheshire, England). Gemcitabine (Gemzar)was purchased from Pfizer (New York City, NY). Serine-, glycine- andfolate-free RPMI 1640 media was custom-ordered from ThermoFisher(Waltham, MA) and supplemented with tissue-culture grade glycine(ThermoFisher) or serine (Sigma-Aldrich), as needed. Other chemicalswere obtained from commercial sources in the highest available purities.

Molecular modeling and computational studies. Molecular modeling wasperformed for all analogs with the human SHMT2 crystal structure (PDB:5V7I) (47) using the induced fit docking protocol of Maestro (48, 49).The ligands were prepared using the Ligprep (50) application of Maestro.The docking protocol was validated by re-docking the co-crystallizedpyranopyrazole ligand (47) into the crystal structure with a RMSD of0.15 Å. The centroid around the pyranopyrazole inhibitor in Chain B wasdefined as the binding site for the compounds. The OPLS 2005 force fieldwas used and amino acid residues within 3 Å from docked poses wereallowed to be optimized using prime refinement (51). The compounds werealso docked into rabbit SHMT1 (PDB: 1LS3) (52) binding sites. Thedocking scores of the analogs are reported in Table S1.

Cell culture and proliferation/protection assays. The HCT116 cell linesincluding the serine hydroxymethyltransferase (SHMT) 1, SHMT2, andmethylene THF dehydrogenase 2 (MTHFD2) knockout (KO) cells werepreviously described (47,53). The H460 cell line was obtained from theAmerican Type Culture Collection (Manassas, VA), whereas the MIA PaCa-2cells were provided by Dr. Yubin Ge (Karmanos Cancer Institute). Celllines were verified by STR analysis by Genetica DNA Laboratories(Burlington, NC). MTXRIIOua^(R)2-4 (i.e. R2) Chinese hamster ovary (CHO)cells were generously provided by Dr. Wayne Flintoff (University ofWestern Ontario) (54). From this parental R2 cell line, human RFC andPCFT were individually transfected to generate the isogenic CHO celllines designated PC43-10 (RFC) and R2/PCFT4 (PCFT) (55-57). Human tumorcell lines were cultured in folate-free RPMI supplemented with 10%dialyzed fetal bovine serum (Sigma-Aldrich), 1% penicillin/streptomycinsolution, 2 mM L-glutamine, and 25 nM leucovorin in a humidifiedatmosphere at 37° C. in the presence of 5% CO₂ and 95% air. The CHO celllines were cultured in alpha-minimal essential medium (alpha-MEM)supplemented with 10% bovine calf serum, 1% penicillin/streptomycinsolution, and 2 mM L-glutamine. Additionally, the transfected CHO celllines (i.e. R2/PCFT4 and PC43-10) were maintained under continuousselection with 1 mg/ml of G418.

For proliferation assays with the CHO cell lines, the cells were treatedwith drugs (0-1 µM) in a 96-well plate (2000 cells/well) inglycine-free, nucleoside-free folate-free RPMI supplemented with 10%dialyzed fetal bovine serum, 1% penicillin/streptomycin, 2 mML-glutamine, and 25 nM leucovorin (final volume 200 µL) over a 96 hincubation period at 37° C. with 5% CO₂. The drugs were dissolved inDMSO; an equivalent amount of DMSO was added to the control (noaddition) samples. To quantify viable cells, the media was removed andplates were washed once with 100 µL Dulbecco’s Phosphate-Buffered Saline(PBS), after which 100 µL PBS and 20 µL Cell Titer-blue (Promega) wereadded. Relative cell numbers were proportional to the fluorescencemeasured with a fluorescence plate reader (590 nm emission, 560 nmexcitation). Background fluorescence (cell-free wells treated with CellTiter-blue) was subtracted and these corrected values were normalized toresults for cells treated in an identical manner without drugs. IC₅₀values, corresponding to the drug concentrations that inhibit growth by50% relative to untreated controls, were generated by fitting a4-parameter logistic regression in Excel.

For proliferation assays of the HCT116, H460, and MIA PaCa2 tumor celllines, the cells were plated in 96-well plates in an identical manner tothe CHO experiments, except that the maximal drug concentration wasincreased to 10 µM. Glycine/nucleoside protection experiments in CHO andtumor cell lines were performed in folate- and glycine-free RPMI1640/10% dialyzed fetal bovine serum supplemented with 25 nM leucovorinwithout additions, or in the presence of adenosine (60 µM), thymidine(10 µM), glycine (130 µM) and/or 5-aminoimidazole-4-carboxamide (AICA)(320 µM). Growth of metabolite-treated cells was normalized to controlstreated with metabolites and vehicle (i.e., DMSO) both singly and incombination. Treatments with all metabolites were performed in parallelon the same plate for a given drug.

Generation of H460 SHMT2 knockdown (H460 SHMT2 KD) cell line. H460 cellswere seeded (2 × 10⁵ cells/well) in 24 well plates containing 1 ml ofculture media (i.e. folate-free RPMI 1640 supplemented with 10% dialyzedfetal bovine serum, 1% penicillin/streptomycin, 2 mM L-glutamine, and 25nM leucovorin). Cells were treated with 4 µg/ml polybrene and 10⁵transducing units of MISSION Lentiviral particles (Sigma-Aldrich)containing shRNA targeting SHMT2 (TRCN0000034805). An additional wellcontained H460 cells without shRNA particles. After 24 hours, the mediawas replaced with fresh culture media including 2 µg/ml puromycin (58)as a selection marker. Once cells were confluent (and non-transducedcells had died), cells were harvested, passaged 3-4 times, then assayedby RT-PCR for SHMT2 knockdown (KD) relative to non-targeted control(NTC) particle-transduced H460 cells (58). To isolate single clones,cells were plated in 100 mm dishes (200 cells/dish) in the presence of 2µg/ml puromycin. Colonies were isolated, and expanded and clonalcultures were assayed for SHMT2 KD via RT-PCR. SHMT2 KD was confirmedvia Western blotting (FIG. 16 ).

Real-time PCR. Cells were harvested from either 60 mm dishes or T25flasks at ~80% confluence and RNAs extracted using TRIzol reagent(Invitrogen, Carlsbad, CA). cDNAs were synthesized with random hexamers,MuLV reverse transcriptase, and RNase inhibitor (Applied Biosystems,Waltham, MA) and purified with a QIAquick PCR Purification Kit (QIAGEN,Valencia, CA). Quantitative real-time RT-PCR was performed using a RocheLightCycler 480 (Roche Diagnostics, Indianapolis, IN) with gene-specificprimers and Universal Probe Library probes (SHMT2-#83, RFC-#32,PCFT-#89, FRα-#65 (Roche Diagnostics)) and transcript levels werenormalized to transcript levels of β-actin and GAPDH. Primer sequencesare available upon request.

Gel electrophoresis and Western blots. H460 wild-type (WT), H460 NTC,H460 SHMT2 KD, HCT116 WT, and HCT116 SHMT2 KO cell lines were cultured,as described above. Cells were plated (1 × 10⁶ cells/dish) in 60 mmdishes and harvested when the cells were ~80% confluent. Cells weredisrupted by sonication with cell debris removed by centrifugation (1800rpm, 5 min, 4° C.). The soluble cell fraction was assayed for proteinconcentration (59) and equal amounts of protein (37 µg) from each samplewere electrophoresed on 10% polyacrylamide gels with SDS (60) andtransferred to polyvinylidene difluoride membranes (ThermoFisher) (61).To detect SHMT2, membranes were incubated for 72 h with rabbitanti-SHMT2 primary antibody (#12762 (53); Cell Signaling Technology,Danvers, MA). The blots were developed by incubating inIRDye800CW-conjugated goat anti-rabbit IgG secondary antibody (LICORBiosciences, Omaha, NE) for 90 min and scanning with an Odyssey infraredimaging system (LICOR Biosciences). Protein loading was normalized toβ-actin using anti-β-actin mouse antibody (Sigma-Aldrich).

Targeted Metabolomics. Targeted metabolomics were performed essentiallyas previously described (47, 53). Briefly, cells (H460, HCT116, MIAPaCa-2) (1 million cells /dish for vehicle-treated samples, 1.5 millioncells/dish for drug-treated samples) were seeded in triplicate 60 mmdishes in 5 ml of folate-free RPMI (contains glycine and unlabeledserine) supplemented with 10% dialyzed fetal bovine serum, 1%penicillin/streptomycin, 2 mM L-glutamine, and 25 nM leucovorin. Cellswere allowed to adhere for 24 h. The media was aspirated and replacedwith culture media (contains 25 nM leucovorin, glycine, and unlabeledserine) and 10 µM AGF291, AGF320 or AGF347, or a comparable volume ofvehicle (DMSO) (with or without 1 mM formate (final concentration)).After 16 h, the cells were washed with PBS (3×), the media was replacedwith folate- and serine-free culture media (containing glycine)supplemented with 10% dialyzed fetal bovine serine, 25 nM leucovorin,and [2,3,3-²H]serine (250 µM), including 10 µM drug or DMSO vehicle. Thecells were incubated for 24 h. All incubations were at 37° C. with 5%CO₂. The media was aspirated, and cells were washed (3×) rapidly (< 30s) with 5 mL ice-cold PBS; metabolism was quickly quenched withmethanol:water (80:20) at -80° C. Cells were allowed to rock on dry icefor 10 min to cover the entire dish with 80:20 methanol:water (at -80°C.), then harvested by scraping and pipetting into 1.5 mL Eppendorftubes. The tubes were centrifuged (4° C., 14000 RPM, 10 min) to fullyextract metabolites into the methanol:water supernatant. The proteinpellet was used for normalization. The supernatants were collected andanalyzed by reversed-phase ion-pairing chromatography coupled withnegative-mode electrospray-ionization high-resolution mass spectrometryon a stand-alone Orbitrap (ThermoFisher Exactive). Raw metabolite valueswere adjusted to correct for normal ion distributions and normalized tototal proteins from the post-extraction pellet by solubilizing with 0.5N NaOH and using the Folin-phenol protein method (13). Values below thelimit of detection were assigned a value of 100 for normalization.Results for drug-treated and SHMT2 KD cells were normalized tovehicle-treated WT ± formate or NTC ± formate samples, as appropriate.

Enzymology

In vivo efficacy trial with MIA PaCa-2 pancreatic cancer xenografts.Methods for in vivo maintenance of MIA PaCa-2 tumor xenografts and drugefficacy evaluations are analogous to those previously described (58,62-67). MIA PaCa-2 human pancreatic cancer cells (5 × 10⁶ cells/flank)were bilaterally implanted subcutaneously with tumor fragments (30-60mg) with a 12-gauge trocar in female NCR SCID mice (NCI AnimalProduction Program). The mice were 10 weeks old on day 0 (tumor implant)with an average body weight of 19 g. For the efficacy trial, the micewere maintained on either a folate-deficient diet from Harlan-Teklad(TD.00434) starting 14 days before subcutaneous tumor implant to ensureserum folate levels would approximate those of humans. A separate cohortof mice was fed a folate-replete control diet from Lab Diet (5021). Micewere supplied with food and water ad libitum. Serum folateconcentrations were monitored prior to tumor implant and post study byLactobacillus casei bioassay (68). The mice in each group(folate-deficient and standard diet) were pooled before unselectivedistribution to the treatment and control groups. Chemotherapy was begun3 days post-tumor implantation with AGF291 (7.75 mg/kg/injection every 6days; total dose of 23.25 mg/kg) or gemcitabine (120 mg/kg/injectionevery 4 days; total dose of 480 mg/kg). The drugs were dissolved in 5%ethanol (v/v), 1% Tween-80 (v/v), and 0.5% NaHCO₃ and were administeredintravenously (0.2 ml/injection). The mice were weighed and tumors weremeasured with a caliper two-to-three times weekly; mice were sacrificedwhen the cumulative tumor burden reached 1500 mg. Tumor weights wereestimated from two-dimensional measurements, where tumor mass (in mg) =(a × b²)/2, and a and b are the tumor length and width in mm,respectively. The tumor masses from both tumors on each mouse were addedtogether, and the total mass per mouse was used for calculations ofanti-tumor activity. Quantitative end-points include: (i) tumor growthdelay [T-C, where T is the median time in days required for thetreatment group tumors to reach a predetermined size (e.g., 1000 mg),and C is the median time in days for the control group tumors to reachthe same size; tumor-free survivors are excluded from thesecalculations]; and (ii) T/C (in percent) when treatment (T) and control(C) groups for the control groups reached 700 mg in size (exponentialgrowth phase). The median value of each group was determined (includingzeros). Mouse weights were monitored as a gauge of drug toxicity.

Statistics: All data shown reflects at least three biological replicatesunless noted otherwise (e.g. targeted metabolomics data, which reflectsthree technical triplicates measured in single experiments). Allstatistical analyses were performed by the Karmanos Cancer InstituteBiostatistics Core. The expression levels were assessed for thenormality assumption. The log₂ transformation was used as all valueswere positive. The statistical tests were carried out using an unpairedt-test. P values were not adjusted for multiple comparisons.

Synthesis of AGF94 and 5-Substituted Pyrrolo[3,2-d]Pyrimidine Compounds

All evaporations were carried out in reduced pressure with a rotaryevaporator. Analytical samples were dried in vacuo in a CHEM-DRY dryingapparatus over P₂O₅ at 50° C. Melting points were determined eitherusing a MEL-TEMP, II melting point apparatus with FLUKE 51 K/Jelectronic thermometer or using an MPA100 OptiMelt automated meltingpoint system and are uncorrected. Nuclear magnetic resonance spectra forproton (¹H NMR) were recorded on the Bruker Avance II 400 (400 MHz) orBruker Avance II 500 (500 MHz) NMR systems with TopSpin processingsoftware. The chemical shift values (δ) are expressed in parts permillion relative to tetramethylsilane as an internal standard.Thin-layer chromatography (TLC) was performed on Whatman® PE SIL G/UV254flexible silica gel plates and the spots were visualized under 254 and365 nm ultraviolet illumination. Proportions of solvents used for TLCare by volume. All analytical samples were homogeneous on TLC in atleast two different solvent systems. Column chromatography was performedon the silica gel (70 to 230 meshes, Fisher Scientific) column. Flashchromatography was carried out on the CombiFlash® Rf systems, modelCOMBIFLASH RF. Pre-packed RediSep® Rf normal-phase flash columns (230 to400 meshes) of diverse sizes were used. The amount (weight) of silicagel for column chromatography was in the range of 50-100 times theamount (weight) of the crude compounds being separated. Elementalanalyses were performed by Atlantic Microlab, Inc., Norcross, GA.Elemental compositions are within ± 0.4% of the calculated values.Fractional moles of water or organic solvents frequently found in someanalytical samples could not be prevented despite 24 to 48 h of dryingin vacuo and were confirmed where possible by their presence in the ¹HNMR spectra. The HPLC measurement was performed using UltiMate 3000UHPLC+ system. Reverse phase HPLC was carried out with a XSelect CSH C18XP, 130 Å, 2.5 µm, 3 mm × 100 mm column. Solvent A: water with 0.1% TFA;Solvent B: acetonitrile. Mass spectrometry m/z determination wasperformed by an Advion Expression-S CMS (a single quadrupole compact MS)controlled by Advion Chems Express 4.0.13.8 software.

AGF 94 (FIG. 9 ) was synthesized as previously described (66).

Synthesis of the target compounds AG291, AGF299, AGF300, AGF318, AGF320,AGF331, AGF347, AGF355 and AGF359 started with a palladium-catalyzedSonogashira coupling of 4-iodobenzoate methyl ester (1a) or4-bromo-thiophene-2-carboxylic acid ethyl ester (1b) or methyl4-bromo-2-fluorobenzoate (1c) with the appropriate alkyne alcohols toafford the appropriate 4-substituted alcohol benzoates 2a-i. Catalytichydrogenation afforded the saturated alcohols 3a-i.(21) The alcohols3a-i were converted to the mesylate derivatives using mesyl chloride andtriethylamine base at 0° C.(69) The mesylate derivatives were notpurified and after workup were converted to their respective iodide 4a-iusing the Finkelstein reaction. The N-alkylation of iodides, 4a-i usingethyl 3-amino-1H-pyrrole-2-carboxylate and sodium hydride underanhydrous conditions afforded the N-5 substituted pyrroles 5a-i.(70)This reaction was incomplete as observed on TLC. Longer reaction timesresulted in decomposition of the product (TLC). The intermediates 5a-icould not be isolated due to presence of multiple spots, even afterrepeated column chromatography. The crude N-substituted pyrroles 5a-iwere directly subjected to condensation with1,3-bis(methoxycarbonyl)-2-methylthiopseudourea with 5 equivalents ofacetic acid as catalyst and MeOH. The hydrolysis of the carbamate groupformed was carried out in situ with aqueous sodium hydroxide at 55° C.to afforded the 2-amino-4-oxo-pyrrolo[3,2-d]pyrimidines 6a-i.(24) Thishydrolysis required higher than room temperature. Performing thehydrolysis at room temperature causes the hydrolysis of the ester, butnot the carbamate (as observed on the ¹H-NMR). However, temperaturesgreater than 70° C. caused degradation of the starting material. Theoptimum temperature for hydrolysis of both ester and carbamate was foundto be 55° C. Conversion of free acids 6a-i to the correspondingL-glutamic acid diethyl esters 7a-i involved conventional peptidecoupling with L-glutamic acid diethyl ester hydrochloride using2-chloro-4,6-dimethoxy-1,3,5-triazine followed by chromatographicpurification to afford the coupled products.(70) Hydrolysis of 7a-i withaqueous NaOH at room temperature, followed by acidification with 1 N HClin the cold, afforded target compounds.

a) PdCl₂, PPh₃, alcohol, TEA, toluene, 1h, 100° C., microwave; b) H₂/Pd,high parr vessel, 24 h, r.t.; c) (i) mesyl chloride, DCM, 0° C., 2 h;(ii) NaI, acetone, 4 h, reflux; d) ethyl3-amino-1H-pyrrole-2-carboxylate, NaH, DMF, 2h, r.t.; e) (i)1,3-bis(methoxycarbonyl)-2-methyl-2-thiopseudourea, MeOH, r.t., 16 h;(ii) NaOMe, MeOH, 16 h, r.t.; (iii) 1 N NaOH, 55° C., 3 h; f) L-glutamicacid diethyl ester hydrochloride, 2-chloro-4,6-dimethoxy-triazine,NMM,DMF, r.t., 12 h; g) 1N NaOH, r.t., 1 h

General procedure for synthesis of 2a-i. In a 20 mL vial for microwavereaction were added a mixture of palladium chloride (71 mg, 0.40 mmol),triphenylphosphine (131 mg, 0.40 mmol), triethylamine (10.1 g, 100mmol), methyl 4-iodobenzoate, 1a (2.21 g, 8.43 mmol) or ethyl5-bromothiophene-2-carboxylate 1b (1.9 g, 8 mmol) or methyl4-bromo-2-fluorobenzoate 1c (2.5 g, 10.73 mmol), and anhydrousacetonitrile (10 mL). To the stirred mixture were added copper(I) iodide(304 mg, 1.60 mmol) and appropriate alkyne alcohol (1.05 equiv), and thevial was sealed and put into the microwave reactor at 100 C for 10 min.Silica gel (5 g) was added, and the solvent was evaporated under reducedpressure. The resulting plug was loaded on to a silica gel column (3.512 cm) and eluted with Hexane followed by 20% EtOAc in Hexane. Thedesired fraction (TLC) was collected, and the solvent was evaporatedunder reduced pressure to afford the target compounds.

Methyl 4-(3-hydroxyprop-1-yn-1-yl)benzoate (2a) Compound 2a wassynthesized using the general method described for the preparation of2a-i using prop-2-yn-1-ol (0.5 ml, 8 mmol), to give 1.3 g of 2a asyellow solid (1.36 g, 85%); TLC Rf= 0.16 (EtOAc:Hexane, 1:2); ¹H-NMR(400 MHz) (Me₂SO-d₆) δ 8.06 - 7.87 (d, J = 8.4 Hz, 2 H, Ar), 7.55 (d, J= 8.4 Hz, 2 H, Ar), 4.30 (s, 1H, exch., -OH), 4.15 (s, 2 H, -CH₂-), 3.81(s, 3 H, -OCH₃). The ¹H-NMR matched the ¹H-NMR reported in theliterature (71).

Methyl 4-(4-hydroxybut-1-yn-1-yl)benzoate (2b) Compound 2b wassynthesized using the general method described for the preparation of2a-i, using but-3-yn-1-ol (0.6 ml, 8 mmol), to give 1.2 g of 2b asyellow solid (1.53 g, 78%); TLC Rf=0.16 (EtOAc:Hexane, 1:2); mp,(72)92.3-94.6° C.; ¹H-NMR (400 MHz) (Me₂SO-d₆) δ 7.90 (d, J = 8.7 Hz, 2 H,Ar), 7.51 (d, J = 8.7 Hz, 2 H, Ar), 4.96 (s, 1 H, exch., -OH), 3.84 (s,3 H, -OCH₃), 3.61 (m, 2 H, -CH₂-), 2.60 (t, J =6.0 Hz, 2 H, -CH₂-). The¹H-NMR matched the ¹H-NMR reported in the literature (73).

Methyl 4-(5-hydroxypent-1-yn-1-yl)benzoate (2c) Compound 2c wassynthesized using the general method described for the preparation of2a-i, using pent-4-yn-1-ol (0.67 ml, 8 mmol), to give 1.34 g of 2c asyellow semi-solid (1.62 g, 88%); TLC Rf 0.16 (EtOAc:Hexane, 1:2); ¹H-NMR(400 MHz) (Me₂SO-d₆) δ 6.50 (d, J = 8.4 Hz, 2 H, Ar), 6.37 (d, J = 8.4Hz, 2 H, Ar), 5.15 (s, 1 H, exch., -OH), 3.61 (s, 3 H, -OCH₃), 3.11 (t,J = 4.9 Hz, 2 H, -CH₂-), 2.64 (t, J = 6.5 Hz, 2 H, -CH₂-), 1.83 - 1.67(m, 2 H, -CH₂-). The ¹H-NMR matched the ¹H-NMR reported in theliterature (74).

Ethyl 5-(3-hydroxyprop-1-yn-1-yl)thiophene-2-carboxylate (2d) Compound2d was synthesized using the general method described for thepreparation of 2a-i, using prop-2-yn-1-ol (0.5 ml, 8 mmol), to give 1.2g of 2d as yellow semi-solid (70%); TLC Rf= 0.11 (EtOAc:Hexane, 1:2);¹H-NMR (400 MHz) (Me₂SO-d₆) δ 7.73 (d, J = 4.0 Hz, 1 H, Ar), 7.36 (d, J= 3.9 Hz, 1 H, Ar), 5.49 (t, J = 6.0 Hz, 1 H, -OH, exch.), 4.37 - 4.28(m, 4 H, -OCH₂ and -CH₂-), 1.29 (t, J = 7.1 Hz, 3 H, -CH₃). Thiscompound was used for the next reaction without furthercharacterization.

Ethyl 5-(4-hydroxybut-1-yn-1-yl)thiophene-2-carboxylatee (2e) Compound388 was synthesized using the general method described for thepreparation of 2a-i, using but-3-yn-1-ol (0.6 ml, 8 mmol), to give 1.1 gof 2e as yellow semi-solid (61%); TLC Rf = 0.11 (EtOAc:Hexane, 1:2);¹H-NMR (400 MHz) (Me₂SO-d₆) δ 7.70 (d, J = 4.0 Hz, 1 H, Ar), 7.28 (d, J= 4.0 Hz, 1 H, Ar), 4.96 (t, J = 5.6 Hz, 1 H, OH, exch.), 3.91-3.81 (m,2 H, -OCH₂), 3.57 (t, J = 6.4 Hz, 2 H, -CH₂), 2.61 (t, J = 6.4 Hz, 2 H,-CH₂), 1.30 (t, J = 7.1 Hz, 3 H, -CH₃). The ¹H-NMR matches the ¹H-NMRreported previously (75).

Ethyl 5-(5-hydroxypent-1-yn-1-yl)thiophene-2-carboxylate (2f) Compound2f was synthesized using the general method described for thepreparation 2a-i, using pent-4-yn-1-ol (0.67 ml, 8 mmol), to give 1.3 gof 2f as yellow semi-solid (68%); TLC Rf=0.11 (EtOAc:Hexane, 1:2);¹H-NMR (400 MHz) (Me₂SO-d₆) δ 7.63 (d, J= 3.8 Hz, 1 H, Ar), 6.95 (d, J =3.8 Hz, 1 H, Ar), 4.44 (t, J = 7.5 Hz, exch., -OH), 4.26 (q, J = 7.0 Hz,2 H, -OCH₂), 2.83 (t, J = 7.6 Hz, 2 H, -CH₂-), 1.66 (p, J = 6.5 Hz, 2 H,-CH₂-), 1.46 (p, J = 6.5 Hz, 2 H, -CH₂-), 1.29-1.24 (m, 3 H, -CH₃). Thiscompound was used for the next reaction without furthercharacterization.

Methyl 2-fluoro-4-(3-hydroxyprop-1-yn-1-yl)benzoate (2 g) Compound 2gwas synthesized using the general method described for the preparationof 2a-i using prop-2-yn-1-ol (1.2 ml, 16.09 mmol), to give 2.02 g of 2gas yellow semi solid (2.02 g, 85%); TLC Rf= 0.3 (EtOAc:Hexane, 1:1); ¹HNMR (400 MHz, Me₂SO-d₆) δ 7.88 (t, J = 7.9 Hz, 1 H, Ar), 7.47 -7.35 (m,2 H, Ar), 5.46 (s, 1 H, exch., -OH), 4.34 (s, 2 H, -CH₂-), 3.86 (s, 3H,-OCH₃). This compound was used for the next reaction without furthercharacterization.

Methyl 2-fluoro-4-(4-hydroxybut-1-yn-1-yl)benzoate (2h) Compound 2h wassynthesized using the general method described for the preparation of2a-i, using but-3-yn-1-ol (0.6 ml, 8 mmol), to give 1.86 g of 2h asyellow solid (1.86 g, 78%); TLC Rf= 0.3 (EtOAc:Hexane, 1:1); mp,(26); ¹HNMR (400 MHz, DMSO-d₆) δ 7.85 (t, J = 8.0 Hz, 1 H, Ar), 7.42 - 7.30 (m,2 H, Ar), 4.98 (t, J = 5.6 Hz, 1 H, exch., -OH), 3.85 (s, 3 H, -OCH₃),3.60 (td, J = 6.7, 5.6 Hz, 2 H, -CH₂-), 2.60 (t, J = 6.7 Hz, 2 H,-CH₂-). This compound was used for the next reaction without furthercharacterization.

Methyl 2-fluoro-4-(5-hydroxypent-1-yn-1-yl)benzoate (2i) Compound 2i wassynthesized using the general method described for the preparation of2a-i, using pent-4-yn-1-ol (1.5 ml, 16.01 mmol), to give 2.23 g of 2i asyellow semi solid (2.23 g, 88%); TLC Rf 0.3 (EtOAc:Hexane, 1:1), ¹H-NMR(400 MHz) (Me₂SO-d₆) δ 7.85 (t, J= 7.9 Hz, 1 H, Ar), 7.38 (d, J = 11.6Hz, 1 H, Ar), 7.33 (d, J = 8.1 Hz, 1 H, Ar), 6.67 (t, J = 3.0 Hz, 0H),4.57 (t, J = 10.4 Hz, 1 H, exch., -OH), 3.85 (s, 3 H, -OCH₃), 3.52 (q,J= 5.8 Hz, 2 H, -CH₂-), 1.69 (q, J= 6.7 Hz, 2 H, -CH₂-). This compoundwas used for the next reaction without further characterization.

General procedure for synthesis of 3a-i. To a Parr flask was added 2a-i,10% palladium on activated carbon (50% w/w), and MeOH (100 mL).Hydrogenation was carried out at 55 psi of H₂ for 4 h. The reactionmixture was filtered through Celite, washed with MeOH (100 mL), andconcentrated under reduced pressure to give crude mixture containing3a-i. Without chromatographic separation, these compounds were used forthe next reaction.

Methyl 4-(3-hydroxypropyl)benzoate (3a) Compound 371 was prepared usingthe general method described for the preparation of 3a-i, from 2a (1.45g, 7.4 mmol) to give 1.2 g (98%) of 3a as a clear oil; TLC Rf=0.16(EtOAc:Hexane, 1:2); ¹H-NMR (400 MHz) (Me₂SO-d₆) 7.93 (d, J = 8.0 Hz, 2H, Ar), 7.55 (d, J = 7.9 Hz, 2 H, Ar), 5.43 (s, 1 H, exch., -OH), 3.85(s, 3 H, -OCH₃), 3.29 (t, J = 7.8 Hz, 2 H, -CH₂-), 2.67 (t, J = 7.8 Hz,2 H, -CH₂-), 1.72 (dt, J = 41.3, 7.4 Hz, 2 H, -CH₂-). This compound wasused for the next reaction without further characterization.

Methyl 4-(4-hydroxybutyl)benzoate (3b) Compound 3b was prepared usingthe general method described for the preparation of 3a-i, from 3a (1.45g, 7.4 mmol) to give 1.1 g (90%) of 3b as a clear oil; TLC Rf= 0.16(EtOAc:Hexane, 1:2); ¹H-NMR (500 MHz) (Me₂SO-d₆) δ 7.88 (d, J = 7.9 Hz,2 H, Ar), 7.34 (d, J = 7.9 Hz, 2 H, Ar), 4.43 (s, 1 H, exch., -OH), 3.83(s, 3 H, -OCH₃), 3.35 - 3.25 (m, 2 H, -CH₂-), 2.68 (q, J = 10.4, 7.9 Hz,2 H, -CH₂-), 1.72 (dtd, J = 49.7, 17.2, 15.4, 9.9 Hz, 4 H, -CH₂-). Thiscompound was used for the next reaction without furthercharacterization. Methyl 4-(5-hydroxypentyl)benzoate (3c) Compound 3cwas prepared using the general method described for the preparation of3a-i, from 2c (1.45 g, 7.4 mmol) to give 1.2 g (98%) of 3c as a clearoil; TLC Rf=0.16 (EtOAc:Hexane, 1:2); ¹H-NMR (400 MHz) (Me₂SO-d₆) δ 7.86(d, J = 7.9 Hz, 2 H, Ar), 7.32 (d, J = 7.9 Hz, 2 H, Ar), 4.36 (s, 1 H,exch., -OH), 3.82 (s, 3 H, -OCH₃), 3.37 (t, J = 6.4 Hz, 2 H, -CH₂-),2.62 (t, J = 7.6 Hz, 2 H, -CH₂-), 1.57 (p, J = 7.7 Hz, 2 H, -CH₂-), 1.43(p, J = 6.6 Hz, 2 H, -CH₂-), 1.29 (ddt, J = 8.6, 6.5, 3.9 Hz, 2 H,-CH₂-). This compound was used for the next reaction without furthercharacterization.

Ethyl 5-(3-hydroxypropyl)thiophene-2-carboxylate (3d) Compound 3d wasprepared using the general method described for the preparation of 3a-i,from 2d (1.1 g, 5.23 mmol) to give 1.0 g (89%) of 3d as a clear oil; TLCRf 0.12 (EtOAc:Hexane, 1:2); ¹H-NMR (400 MHz) (Me₂SOd₆) δ 7.63 (t, J =6.1 Hz, 1 H, Ar), 6.94 (d, J = 6.1 Hz, 1 H, Ar), 4.44 (t, J = 5.1 Hz, 1H, exch., -OH), 4.26 (p, J = 8.3, 7.1 Hz, 2 H, -OCH₂-), 2.83 (t, J = 7.6Hz, 2 H, -CH₂-), 1.66 (p, J = 7.6 Hz, 2 H, -CH₂-), 1.46 (p, J = 6.8 Hz,2 H, -CH₂-), 1.27 (t, J = 7.0 Hz, 3 H, -CH₃). This compound was used forthe next reaction without further characterization.

Ethyl 5-(4-hydroxybutyl)thiophene-2-carboxylate (3e) Compound 3e wasprepared using the general method described for the preparation of 3a-i,from 2e (1.2 g, 5.35 mmol) to give 1.0 g (82%) of 3e as a clear oil; TLCRf= 0.12 (EtOAc:Hexane, 1:2); ¹H-NMR (400 MHz) (Me₂SO-d₆) δ 7.63 (d, J =3.7 Hz, 1 H, Ar), 6.95 (d, J = 3.8 Hz, 1 H, Ar), 4.38 (s, 1 H, exch.,-OH), 4.23-4.20 (m, 2 H, -OCH₂-), 2.84 (q, J = 9.6, 7.5 Hz, 2 H, -CH₂-),1.63 (p, J = 7.5 Hz, 3 H, -CH₃), 1.56 -1.39 (m, 2 H, -CH₂-), 1.24-1.50(m, 4 H, -CH₂-). The ¹H-NMR matches ¹H-NMR of the reported compound(29).

Ethyl 5-(5-hydroxypentyl)thiophene-2-carboxylate (3f) Compound 3f wasprepared using the general method described for the preparation of 3a-i,from 2f (1.1 g, 4.62 mmol) to give 1.0 g (89%) of 3f as a clear oil; TLCRf=0.12 (EtOAc:Hexane, 1:2); ¹H-NMR (400 MHz) (Me₂SO-d₆) δ 7.63 (d, J =3.7 Hz, 1 H, Ar), 6.95 (d, J = 3.8 Hz, 1 H, Ar), 4.36 (s, 1 H, exch.,-OH), 4.25 (q, J = 7.0 Hz, 2 H, -OCH₂-), 3.38 (t, J = 6.3 Hz, 2 H,-CH₂-), 2.83 (t, J = 7.4 Hz, 2 H, -CH₂-), 1.63 (p, J = 7.5 Hz, 2 H,-CH₂-), 1.44 (p, J = 6.6 Hz, 2 H, -CH₂-), 1.39 - 1.30 (m, 2 H, -CH₂-),1.28 (t, J = 7.0 Hz, 3 H, -CH₃). This compound was used for the nextreaction without further characterization.

Methyl 2-fluoro-4-(3-hydroxypropyl)benzoate (3g) Compound 3g wasprepared using the general method described for the preparation of3a-i,from 2g (2.02 g, 9.7 mmol) to give 1.98 g (98%) of 3g as a clear oil;TLC Rf= 0.3 (EtOAc:Hexane, 1:1); ¹H-NMR (400 MHz) (Me₂SO-d₆) δ 7.81 (t,J = 7.8 Hz, 1 H, Ar), 7.24 - 7.14 (m, 2 H, Ar), 4.55 (s, 1H, exch.,-OH), 3.84 (s, 3 H, -OCH₃), 3.41 (d, J = 6.3 Hz, 2 H, -CH₂-), 2.73 -2.64 (m, 2 H, -CH₂-), 1.78 - 1.68 (m, 2 H, -CH₂-).This compound was usedfor the next reaction without further characterization.

Methyl 2-fluoro-4-(4-hydroxybutyl)benzoate (3h) Compound 3h was preparedusing the general method described for the preparation of 3a-i, from 3h(1.86 g, 8.4 mmol) to give 1.68 g (90%) of 3b as a clear oil; TLC Rf=0.3 (EtOAc:Hexane, 1:1); ¹H-NMR (500 MHz) (Me₂SO-d₆) δ 7.80 (t, J = 7.9Hz, 1 H, Ar), 7.22 - 7.14 (m, 2 H, Ar), 4.39 (s, 1 H, exch., -OH), 3.84(s, 3 H, -OCH₃), 3.40 (d, J= 11.7 Hz, 2 H, -CH₂-), 2.65 (t, J = 7.7 Hz,2 H, -CH₂-), 1.66 - 1.55 (m, 2 H, -CH₂-), 1.42 (dt, J= 13.4, 6.5 Hz, 2H, -CH₂-). This compound was used for the next reaction without furthercharacterization.

Methyl 2-fluoro-4-(5-hydroxypentyl)benzoate (3i) Compound 3i wasprepared using the general method described for the preparation of 3a-i,from 2i (2.23 g, 9.44 mmol) to give 2.23 g (98%) of 3c as a clear oil;TLC Rf= 0.3 (EtOAc:Hexane, 1:1); ¹H-NMR (400 MHz) (Me₂SO-d₆) ¹H NMR (400MHz, DMSO-d6) δ 7.80 (t, J = 7.9 Hz, 1 H, Ar), 7.24 - 7.13 (m, 2 H, Ar),4.37 (s, 1 H, exch., -OH), 3.84 (s, 3 H, -OCH₃), 3.37 (t, J = 6.4 Hz, 2H, -CH₂-), 2.69 - 2.60 (m, 2 H, -CH₂-), 1.58 (p, J = 7.6 Hz, 2 H,-CH₂-), 1.44 (dd, J = 14.2, 7.4 Hz, 2 H, -CH₂-), 1.35 - 1.23 (m, 2 H,-CH₂-). This compound was used for the next reaction without furthercharacterization.

General procedure for synthesis of 4a-i. To the alcohols 3a-i, was addedtriethylamine (1 equivalent) and dichloromethane (25 mL). The reactionwas cooled to 0° C. and purged with nitrogen gas. Under anhydrousconditions, methanesulfonyl chloride (1.05 equivalent) was addeddropwise over 30 minutes. The reaction was stirred at room temperaturefor 2 hours and the reaction was added into sodium bicarbonate solution(25 mL). The water layer was washed thrice with dichloromethane (100mL). The dichloromethane was evaporated to obtain a semi-solid product.To the intermediate in acetone, sodium iodide (1 equivalent) was addedand refluxed for 8 hours. The reaction mixture was filtered. Thefiltrate was evaporated to obtain 4a-i.

Methyl 4-(3-iodopropyl)benzoate (4a) Compound 4a was prepared using thegeneral method described for the preparation of 4a-i, from 3a (1 g, 4.5mmol), methanesulfonyl chloride (0.35 mL, 4.5 mmol) and triethylamine(0.62 mL, 4.5 mmol) to form the intermediate. To this sodium iodide wasadded and the procedure was followed to give 0.9 g (72%) of 4a as aclear oil; TLC Rf= 0.63 (EtOAc:Hexane, 1:2); ¹H-NMR (400 MHz) (Me₂SO-d₆)δ 7.95 - 7.83 (d, J = 8.0 Hz, 2 H, Ar), 7.36 (d, J = 8.0 Hz, 2 H, Ar),3.84 (s, 3 H, -OCH₃), 3.24 (t, J = 6.8 Hz, 2 H, -CH₂-), 2.74 (t, J = 7.5Hz, 2 H, -CH₂-), 2.07 (p, J = 7.0 Hz, 2 H, -CH₂-).

This compound was used for the next reaction without furthercharacterization.

Methyl 4-(4-iodobutyl)benzoate (4b) Compound 4b was prepared using thegeneral method described for the preparation of 4a-i, from 3b (1 g, 4.5mmol), methanesulfonyl chloride (0.35 mL, 4.5 mmol) and triethylamine(0.62 mL, 4.5 mmol) to form the intermediate. To this sodium iodide wasadded and the procedure was followed to give 1.0 g (80%) of 4b as aclear oil; TLC Rf= 0.63 (EtOAc:Hexane, 1:2); ¹H-NMR (500 MHz) (Me₂SO-d₆)δ 7.86 (d, J = 8.2 Hz, 2 H, Ar), 7.31 (d, J = 8.3 Hz, 2 H, Ar), 4.55 (t,J = 5.1 Hz, 2 H, -CH₂-), 3.82 (s, 3 H, -OCH₃), 3.41 (t, J = 6.4, 2 H,-CH₂-), 2.65 (t, J = 6.4, 2 H, -CH₂-), 1.78 - 1.66 (m, 2 H, -CH₂-),1.47-1.40 (m, 2 H, -CH₂). This compound was used for the next reactionwithout further characterization.

Methyl 4-(5-iodopentyl)benzoate (4c) Compound 4c was prepared using thegeneral method described for the preparation of 4a-i, from 3c (1 g, 4.5mmol), methanesulfonyl chloride (0.35 mL, 4.5 mmol) and triethylamine(0.62 mL, 4.5 mmol) to form the intermediate. To this sodium iodide wasadded and the procedure was followed to give 1.05 g (85%) as 4c clearoil; TLC Rf= 0.63 (EtOAc:Hexane, 1:2); ¹H-NMR (400 MHz) (Me₂SO-d₆) δ7.87 (d, J = 8.2 Hz, 2 H, Ar), 7.31 (d, J = 8.3 Hz, 2 H, Ar), 3.82 (s, 3H, -OCH₃), 3.23 (t, J = 6.9 Hz, 2 H, -CH₂-), 2.62 (t, J = 7.7 Hz, 2 H,-CH₂-), 1.76 (p, J = 7.0 Hz, 2 H, -CH₂-), 1.57 (tt, J = 9.2, 6.9 Hz, 2H, -CH₂-), 1.40 -1.29 (m, 2 H, -CH₂-). This compound was used for thenext reaction without further characterization.

Ethyl 5-(3-iodopropyl)thiophene-2-carboxylate (4d) Compound 4d wasprepared using the general method described for the preparation of 4a-i,from 3d (0.9 g, 4.5 mmol), methanesulfonyl chloride (0.35 mL, 4.5 mmol)and triethylamine (0.62 mL, 4.5 mmol) to form the intermediate. To thissodium iodide was added and the procedure was followed to give 0.85 g(61%) of 4d as a clear oil; TLC Rf= 0.63 (EtOAc:Hexane, 1:2); ¹H-NMR(400 MHz) (Me₂SO-d₆) δ 7.63 (t, J = 4.6 Hz, 1 H, Ar), 6.97 (d, J = 3.7Hz, 1 H, Ar), 4.25 (q, J = 7.1 Hz, 2 H, -CH₂-), 3.26 (t, J = 6.8 Hz, 2H, -CH₂-), 2.93 (q, J = 9.0, 7.4 Hz, 2 H, -CH₂-), 2.10 (p, J = 6.9 Hz, 2H, -CH₂-), 1.28 (t, J = 7.1 Hz, 3 H, -CH₃). This compound was used forthe next reaction without further characterization.

Ethyl 5-(4-iodobutyl)thiophene-2-carboxylate (4e) Compound 4e wasprepared using the general method described for the preparation of 4a-i,from 3e (0.95 g, 4.5 mmol), methanesulfonyl chloride (0.35 mL, 4.5 mmol)and triethylamine (0.62 mL, 4.5 mmol) to form the intermediate. To thissodium iodide was added and the procedure was followed to give 0.9 g(63%) of 4e as a clear oil; TLC Rf= 0.63 (EtOAc:Hexane, 1:2); ¹H-NMR(400 MHz) (Me₂SOd₆) δ 7.64 (d, J = 3.8 Hz, 1 H, Ar), 6.97 (d, J = 3.8Hz, 1 H, Ar), 4.26 (q, J = 7.1 Hz, 2 H, -CH₂-), 3.46 - 3.24 (m, 2 H,-CH₂-), 2.87 (t, J = 7.3 Hz, 2 H, -CH₂-), 1.93 - 1.64 (m, 4 H, -CH₂-),1.28 (t, J = 7.1 Hz, 3 H, -OCH₃). This compound was used for the nextreaction without further characterization.

Ethyl 5-(5-iodopentyl)thiophene-2-carboxylate (4f) Compound 395 wasprepared using the general method described for the preparation of 4a-i,from 3f (1 g, 4.38 mmol), methanesulfonyl chloride (0.35 mL, 4.5 mmol)and triethylamine (0.62 mL, 4.5 mmol) to form the intermediate. To thissodium iodide was added and the procedure was followed to give 0.95 g(64%) of 4f as a clear oil; TLC Rf= 0.63 (EtOAc:Hexane, 1:2); ¹H-NMR(400 MHz) (Me₂SO-d₆) δ 7.60 (t, J = 4.6 Hz, 1 H, Ar), 7.21 (d, J = 3.7Hz, 1 H, Ar), 4.22 (q, J = 7.1 Hz, 2 H, -CH₂-), 3.35 - 3.27 (m, 2 H,-CH₂-), 3.12 (tt, J = 9.3, 5.2 Hz, 2 H, -CH₂-), 2.57 (t, J = 6.8 Hz, 2H, -CH₂-), 1.97 (q, J = 7.1 Hz, 2 H, -CH₂-), 1.29 - 1.12 (m, 5 H, -CH₂-and -CH₃). This compound was used for the next reaction without furthercharacterization.

Methyl 2-fluoro-4-(3-iodopropyl)benzoate (4g) Compound 4g was preparedusing the general method described for the preparation of 4a-i, from 3g(1.98 g, 9.33 mmol), methanesulfonyl chloride (0.96 mL, 12.4 mmol) andtriethylamine (2 mL, 14 mmol) to form the intermediate. To this sodiumiodide was added and the procedure was followed to give 2.17 g (72%) of4a as a clear oil; TLC Rf= 0.8 (EtOAc:Hexane, 1:1); ¹H-NMR (400 MHz)(Me₂SO-d₆) δ 7.82 (t, J= 7.9 Hz, 1 H. Ar), 7.28 - 7.16 (m, 2 H, Ar),3.84 (s, 3 H, -OCH₃), 3.24 (t, J = 6.9 Hz, 2 H, -CH₂-), 2.79 - 2.70 (m,2 H, -CH₂-), 2.13 - 2.04 (m, 2 H, -CH₂-). This compound was used for thenext reaction without further characterization.

Methyl 2-fluoro-4-(4-iodobutyl)benzoate (4h) Compound 4h was preparedusing the general method described for the preparation of 4a-i, from 3h(1.7 g, 7.51 mmol), methanesulfonyl chloride (0.77 mL, 9.99 mmol) andtriethylamine (1.6 mL, 11.27 mmol) to form the intermediate. To thissodium iodide was added and the procedure was followed to give 2.02 g(80%) of 4b as a clear oil; TLC Rf= 0.8 (EtOAc:Hexane, 1:1); ¹H-NMR (500MHz) (Me₂SO-d₆) δ 7.86 - 7.77 (m, 1 H, Ar), 7.25 - 7.13 (m, 2 H, Ar),3.84 (s, 3 H, Ar), 3.32 - 3.25 (m, 2 H, -CH₂-), 2.67 (t, J = 7.4 Hz, 2H, -CH₂-), 1.82 - 1.72 (m, 2 H, -CH₂-), 1.70 - 1.6 (m, 2 H, -CH₂-). Thiscompound was used for the next reaction without furthercharacterization.

Methyl 2-fluoro-4-(5-iodopentyl)benzoate (4i) Compound 4i was preparedusing the general method described for the preparation of 4a-i, from 3i(2.23 g, 9.28 mmol), methanesulfonyl chloride (0.96 mL, 12.34 mmol) andtriethylamine (1.9 mL, 13.92 mmol) to form the intermediate. To thissodium iodide was added and the procedure was followed to give 2.75 g(85%) as 4c clear oil; TLC Rf= 0.8 (EtOAc:Hexane, 1:1); ¹H-NMR (400 MHz)(Me₂SO-d₆) δ 7.81 (t, J= 7.9 Hz, 1 H, Ar), 7.30 - 7.18 (m, 2 H, Ar),3.84 (s, 3 H, -OCH₃), 3.28 (t, J= 6.9 Hz, 2 H, -CH₂-), 2.66 (t, J = 7.6Hz, 2 H, -CH₂-), 1.83 - 1.73 (m, 2 H, -CH₂-), 1.67 - 1.58 (m, 2 H,-CH₂-), 1.37 (q, J= 7.8 Hz, 2 H, -CH₂-). This compound was used for thenext reaction without further characterization.

General procedure for synthesis of 6a-i. To a solution of ethyl3-amino-1H-pyrrole-2-carboxylate hydrochloride (0.5 g, 3.24 mmol) in dryDMF (10 mL) was added slowly NaH (0.17 g, 7.1 mmol) under nitrogen atroom temperature. The resulting mixture was stirred for about 15 minwhen there was no more gas produced, and then appropriate iodide (1equivalent) was added. The reaction mixture was stirred at roomtemperature for 4 h, and DMF was evaporated at elevated temperature tooffer a gummy residue, which was used for the next step withoutpurification. The gummy residue was dissolved in MeOH (10 mL), and1,3-bis(methoxycarbonyl)-2-methyl-2- thiopseudourea (0.7 g, 3.3 mmol)was added followed by AcOH (1.0 g, 15 mmol). The mixture was stirred atroom temperature overnight and became a thick paste. NaOMe in MeOH (25%)(7 mL, 22 mmol) was added, and stirring was continued at roomtemperature overnight. The mixture was neutralized with AcOH, and themethanol was removed under reduced pressure. To the residue was addedwater (20 mL), and the pH value was adjusted to 10-11 by adding NH₃ ·H₂O. The solid was collected by filtration and washed well with water.The resulting solid was added to 1 N NaOH (2 mL), and the mixture washeated at 55° C. for 3 h. The mixture was cooled and acidified using 1 Nhydrochloric acid. The precipitate was collected and dried under reducedpressure overnight to obtain 6a-i.

General procedure for synthesis of 7a-i. To a solution of 6a-i inanhydrous DMF (10 mL) was added N-methylmorpholine (73 mg, 0.72 mmol)and 2-chloro-4,6-dimethoxy-1,3,5-triazine (127 mg, 0.72 mmol). Theresulting mixture was stirred at room temperature for 2 h. To thismixture was added N-methylmorpholine (73 mg, 0.72 mmol) and L-glutamatediethyl ester hydrochloride (144 mg, 0.6 mmol). The reaction mixture wasstirred for an additional 4 h at room temperature. Silica gel (400 mg)was then added, and the solvent was evaporated under reduced pressure.The resulting plug was loaded on to a silica gel column with 5% MeOH inCHCl₃ as the eluent. Fractions that showed the desired spot (TLC) werepooled and the solvent evaporated to dryness to afford compounds 7a-i.

General method for synthesis of target compounds. To a solution of 7a-i,was added 4 mL methanol and 2 mL of 1 N sodium hydroxide solution. Thereaction mixture was stirred for 1 hour at room temperature and thedisappearance of the starting material was spotted with TLC. The mixturewas acidified to pH 2-3 using 1 N hydrochloric acid to obtain targetcompounds as precipitate on filtration.

Diethyl(4-(3-(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)propyl)benzoyl-L-glutamate (7a) Using the general method for synthesis ofcompounds 6a-i, 5a (1.1 g, 3.62 mmol) was used to obtain 6a (0.3 g, 30%)as a white solid. Using the general method for synthesis of compounds7a-i, 6a (0.15 g, 0.48 mmol) was used to obtain 7a (0.18 g, 75%) as agreyish brown solid; TLC Rf= 0.23 (MeOH:CHCl₃:NH₄OH, 1:10:0.5); ¹H-NMR(400 MHz) (Me₂SO-d₆) δ 10.49 (s, 1 H, exch., -NH), 8.67 (d, J = 7.4 Hz,1 H, exch., -NH), 7.80 (d, J = 8.0 Hz, 2 H, Ar), 7.29 (d, J = 8.0 Hz, 2H, Ar), 7.21 (d, J = 2.6 Hz, 1 H, Ar), 5.91 (d, J = 2.7 Hz, 1 H, Ar),5.81 (s, 2 H, exch., 2-NH₂), 4.42 (d, J = 7.4 Hz, 1 H, -CH), 4.30 - 4.20(m, 2 H, -CH₂-), 4.17 - 3.94 (m, 4 H, -CH₂-), 2.65 - 2.54 (m, 2 H,-CH₂-), 2.44 (t, J = 7.4 Hz, 2 H, -CH₂-), 2.22 - 1.86 (m, 4 H, -CH₂-),1.35 - 1.09 (m, 6 H, -CH₃). Anal. Calcd. C₂₅H₃₁ N₅O₆: C, 60.35; H, 6.28;N, 14.08; O, 19.29. Found: C, 60.03; H, 6.17; N, 13.76.

Diethyl(4-(4-(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)butyl)benzoyl)-L-glutamate (7b) Using the general method for synthesis ofcompounds 6a-i, 5b (1.1 g, 3.46 mmol) was used to obtain 6b (0.25 g,25%) as a white solid; TLC Rf= 0.0 (MeOH:CHCl₃:HCl, 1:5:0.5); ¹H-NMR(400 MHz) (Me₂SO-d₆) δ 10.50 (s, br, exch., -COOH), 7.81 (d, J = 7.9 Hz,2 H, Ar), 7.21 (d, J = 8.0 Hz, 2 H, Ar), 7.18 (d, J = 2.9 Hz, 1 H, Ar),5.92 (s, 2 H, exch., 2-NH₂), 5.87 (d, J = 2.7 Hz, 1 H, Ar), 4.25 (t, J =6.8 Hz, 2 H, -CH₂-), 2.61 (t, J = 7.7 Hz, 2 H, -CH₂-), 1.73 (p, J = 7.8Hz, 2 H, -CH₂-), 1.48 (p, J = 7.8 Hz, 2 H, -CH₂-). The melting pointassessment suggested impurities and hence this compound was used for thenext reaction without further characterization. Using the general methodfor synthesis of compounds 7a-i, 6b (0.15 g, 0.46 mmol) was used toobtain 7b (0.1 g, 43%) as a brown solid; TLC Rf= 0.23 (MeOH:CHCl₃:NH₄OH,1:10:0.5); ¹H-NMR (400 MHz) (Me₂SO-d₆) δ 10.44 (s, 1 H, exch., -NH),8.64 (d, J = 7.4 Hz, 1 H, exch., -NH), 7.77 (d, J = 8.2 Hz, 2 H, Ar),7.26 (d, J = 8.2 Hz, 2 H, Ar), 7.19 (d, J = 2.9 Hz, 1 H, Ar), 5.88 (s, J= 2.9 Hz, 1 H, Ar), 5.76 (s, 2 H, exch., 2-NH₂), 4.45-4.41 (m, 1 H,-CH), 4.25 (t, J = 6.8 Hz, 2 H, -CH₂-), 4.18 - 4.00 (m, 4 H, -CH₂-),2.62 (t, J = 7.6 Hz, 2 H, -CH₂-), 2.44 (t, J = 7.5 Hz, 2 H, -CH₂-),2.28 - 1.89 (m, 2 H, -CH₂-), 1.87 - 1.62 (m, 2 H, -CH₂-), 1.60 - 1.40(m, 2 H, -CH₂-), 1.18 (dt, J = 9.9, 7.0 Hz, 6 H, -CH₃). Anal. Calcd.C₂₆H₃₃N₅O₆ 0.05 CHCl₃: C, 61.04; H, 6.50; N, 13.69; O, 18.76. Found: C,60.57; H, 6.44; N, 13.28.

Diethyl(4-(5-(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)pentyl)benzoyl)-L-glutamate (7c) Using the general method for synthesis ofcompounds 6a-i, 5c (1.2 g, 3.61 mmol) was used to obtain 6c (0.32 g,29%) as a white solid; TLC Rf= 0.0 (MeOH:CHCl₃:HCl, 1:5:0.5); ¹H-NMR(400 MHz) (Me₂SO-d₆) δ 12.74 (s, 1 H, exch., -COOH), 8.14 (s, 2 H,exch., 2-NH₂), 7.83 (d, J = 7.9 Hz, 2 H, Ar), 7.45 (d, J = 2.9 Hz, 1 H,Ar), 7.28 (d, J = 8.0 Hz, 2 H, Ar), 6.13 (d, J = 2.9 Hz, 1 H, Ar), 4.29(t, J = 6.9 Hz, 2 H, -CH₂-), 3.37 (t, J = 6.4 Hz, 2 H, -CH₂-), 2.64 (t,J = 7.8 Hz, 2 H, -CH₂-), 1.79 - 1.70 (m, 2 H, -CH₂-), 1.54 - 1.45 (m, 2H, -CH₂-). The melting point assessment suggested impurities and hencethis compound was used for the next reaction without furthercharacterization. Using the general method for synthesis of compounds7a-i, 6c (0.15 g, 0.44 mmol) was used to obtain 7c (0.11 g, 47.50%) as agrey solid TLC Rf= 0.23 (MeOH:CHCl₃:NH₄OH, 1:10:0.5); ¹H-NMR (400 MHz)(Me₂SO-d₆) δ 10.45 (s, 1 H, exch., -NH), 8.66 (d, J = 7.6 Hz, 1 H,exch., -NH), 7.77 (d, J = 8.0 Hz, 2 H, Ar), 7.27 (d, J = 8.0 Hz, 2 H,Ar), 7.16 (d, J = 2.7 Hz, 1 H, Ar), 5.87 (d, J = 2.8 Hz, 1 H, -Ar), 5.75(s, 2 H, exch., -NH₂), 4.41 (d, J = 13.0 Hz, 1 H, -CH), 4.19 (t, J = 6.9Hz, 2 H, -CH₂-), 4.14 - 3.96 (m, 4 H, -CH₂), 2.59 (t, J = 7.6 Hz, 2 H,-CH₂-), 2.43 (t, J = 7.4 Hz, 2 H, -CH₂-), 2.20 - 1.92 (m, 2 H, -CH₂-),1.81 - 1.65 (m, 2 H, -CH₂-), 1.63 - 1.51 (m, 2 H, -CH₂-), 1.22-1.11 (m,8 H, -CH₂-and -CH₃). Anal. Calcd. for C₂₇H₃₅N₅O₆ 0.24 H₂O: C, 61.70; H,6.71; N, 13.32. Found: C, 61.20; H, 6.763; N, 13.13.

Diethyl(5-(3-(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)propyl)thiophene-2-carbonyl)-L-glutamate (7d) Using the general method forsynthesis of compounds 6a-i, 5d (1.0 g, 2.97 mmol) was used to obtain 6d(0.18 g, 19%) as a white solid; TLC Rf= 0.0 (MeOH:CHCl₃:HCl, 1:5:0.5);¹H-NMR (400 MHz) (Me₂SO-d₆) δ 11.22 (s, br, 1 H, exch., -COOH), 7.49 (d,J = 3.6 Hz, 1 H, Ar), 7.19 (d, J = 3.0 Hz, 1 H, Ar), 6.87 (d, J = 3.8Hz, 1 H, Ar), 6.00 (s, 2H, exch., 2-NH₂), 5.94 (d, J = 3.0 Hz, 1 H, Ar),4.25 (t, J = 6.8 Hz, 2 H, -CH₂), 2.70 (t, J = 7.8 Hz, 2 H, -CH₂-), 2.06(t, J = 7.6 Hz, 2 H, -CH₂-). The melting point assessment suggestedimpurities and hence this compound was used for the next reactionwithout further characterization. Using the general method for synthesisof compounds 7a-i, 6d (0.15 g, 0.47 mmol) was used to obtain 7d (0.125g, 53%) as a grey semi-solid; TLC Rf= 0.23 (MeOH:CHCl₃:NH₄OH, 1:10:0.5);¹H-NMR (400 MHz) (Me₂SO-d₆) δ 10.44 (s, 1 H, exch., -NH), 8.62 (d, J =7.5 Hz, 1 H, exch., -NH), 7.69 (d, J = 3.8 Hz, 1 H, Ar), 7.20 (d, J =2.9 Hz, 1 H, Ar), 6.91 (d, J = 3.8 Hz, 1 H, Ar), 5.91 (d, J = 2.9 Hz, 1H, Ar), 5.77 (s, 2 H, exch., 2-NH₂), 4.38 (dt, J = 9.4, 5.9 Hz, 1 H,-CH), 4.27 (t, J = 6.8 Hz, 2 H, -CH₂-), 4.08 (dq, J = 23.8, 7.0 Hz, 4 H,-CH₂-), 2.72 (t, J = 7.9 Hz, 2 H, -CH₂-), 2.42 (t, J = 7.5 Hz, 2 H,-CH₂-), ), 2.10 (q, J = 7.2 Hz, 2 H, -CH₂-), 1.97 (ddd, J = 16.7, 14.0,7.6 Hz, 2 H, -CH₂-), 1.18 (dt, J = 9.0, 7.1 Hz, 6 H, -CH₃). Thiscompound was used for the next reaction without furthercharacterization.

Diethyl(5-(4-(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)butyl)thiophene-2-carbonyl)-L-glutamate (7e) Using the general method forsynthesis of compounds 6a-i, 5e (1.0 g, 2.97 mmol) was used to obtain 6e(0.20 g, 20%) as a white solid. Using the general method for synthesisof compounds 7a-i, 6e (0.18 g, 0.54 mmol) was used to obtain 7e (0.1 g,37%) as a brown semi- solid; TLC Rf= 0.23 (MeOH:CHCl₃:NH₄OH, 1:10:0.5);¹H-NMR (400 MHz) (Me₂SO-d₆) δ 10.45 (s, 1 H, exch., -NH), 8.63 (d, J =7.7 Hz, 1 H, exch., -NH), 7.67 (d, J = 3.8 Hz, 1 H, Ar), 7.20 (d, J =2.5 Hz, 1 H, Ar), 6.85 (d, J = 3.9 Hz, 1 H, Ar), 5.88 (d, J = 2.9 Hz, 1H, Ar), 5.76 (s, 2 H, exch., 2-NH₂), 4.3-4.45 (m, 1 H, -CH), 4.26 (t, J= 6.8 Hz, 2 H, -CH₂-), 4.07 (dq, J = 22.7, 7.2 Hz, 4 H, -CH₂-), 3.46 -3.24 (m, 2 H, -CH₂-), 2.79 (t, J = 7.4 Hz, 2 H, -CH₂-), 2.42 (t, J = 7.4Hz, 2 H, -CH₂-), 1.76 (t, J = 7.6 Hz, 2 H, -CH₂-), 1.51 (t, J = 7.6 Hz,2 H, -CH₂-), 1.17 (dt, J = 9.5, 7.1 Hz, 6 H, -CH₃). This compound wasused for the next reaction without further characterization.

Diethyl (5-(5-(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)pentyl) thiophene-2-carbonyl)-L-glutamate (7f)Using the general method for synthesis of compounds 6a-i, 5f (2.0 g,5.74 mmol) was used to obtain 6f (0.34 g, 30%) as a white solid. Usingthe general method for synthesis of compounds 7a-i, 6f (0.34 g, 1.03mmol) was used to obtain 7f (0.11 g, 72%) as a grey semi-solid; TLC Rf=0.23 (MeOH:CHCl₃:NH₄OH, 1:10:0.5); ¹H-NMR (400 MHz) (Me₂SO-d₆) δ 10.69(s, 1H, exch., -NH-), 8.65 (d, J = 7.5 Hz, 1 H, exch., -NH), 7.69 (d, J= 3.8 Hz, 1 H, Ar), 7.16 (d, J = 2.9 Hz, 1 H, Ar), 6.85 (d, J = 3.7 Hz,1 H, Ar), 6.12 - 5.71 (m, 3 H, Ar (1 H) and 2-NH₂ (2 H, exch.)), 4.41(d, J = 5.6 Hz, 1 H, -CH), 4.21 (t, J = 6.8 Hz, 2 H, -CH₂-), 4.11 (q, J= 7.1 Hz, 2 H, -CH₂-), 4.04 (q, J = 7.1 Hz, 2 H, -CH₂-), 2.75 (t, J =7.4 Hz, 2 H, -CH₂-), 2.42 (d, J = 7.5 Hz, 2 H, -CH₂-), 2.21 - 1.89 (m, 2H, -CH₂-), 1.73 (t, J = 7.4 Hz, 2 H, -CH₂-), 1.60 (t, J = 7.6 Hz, 2 H,-CH₂-), 1.16 (m, 8 H, -CH₂- and -CH₃). This compound was used for thenext reaction without further characterization.

Diethyl(4-(3-(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)propyl)-2-fluorobenzoyl)-L-glutamate(7g) Using the general method for synthesis of compounds 6a-i, 5g (1.1g, 3.62 mmol) was used to obtain 6g (0.3 g, 30%) as a white solid; TLCRf= 0.0 (MeOH:CHCl₃:HCl, 1:5:0.5); mp, 121.8-156.3° C.; ¹H-NMR (500 MHz)(Me₂SO-d₆) δ 7.77 (t, J = 7.9 Hz, 1 H, Ar), 7.24 (d, J = 2.9 Hz, 1 H,Ar), 7.16 - 7.10 (m, 2 H, Ar), 6.09 (s, 2 H, exch., 2-NH₂), 5.94 (d, J=2.8 Hz, 1 H, Ar), 4.26 (t, J= 7.0 Hz, 2 H, -CH₂-), 2.59 (dd, J= 8.9, 6.8Hz, 2 H, -CH₂-), 2.09 - 2.02 (m, 2 H, -CH₂-).Using the general methodfor synthesis of compounds 7a-i, 6g (0.15 g, 0.48 mmol) was used toobtain 7 g (0.2 g, 75%) as a brown solid; TLC Rf= 0.3 (MeOH:CHCl₃:NH₄OH,1:10:0.5); ¹H-NMR (500 MHz) (Me₂SO-d₆) δ 11.06 (s, 1 H, exch., -NH),8.58 (dd, J = 7.6, 2.0 Hz, 1 H, Ar), 7.51 (t, J = 7.8 Hz, 1 H, Ar), 7.21(d, J = 2.9 Hz, 1 H, Ar), 7.18 - 7.08 (m, 2 H, Ar), 5.91 (d, J = 2.8 Hz,1 H, Ar), 5.80 (s, 2H, exch., 2-NH₂), 4.45 -4.37 (m, 1 H, -CH), 4.25 (t,J= 7.0 Hz, 2 H, -CH₂-), 4.12 (qq, J= 7.0, 3.7 Hz, 2 H, -CH₂-), 4.05 (q,J= 7.1 Hz, 2 H, Ar), 3.86 (s, 2 H, Ar), 2.58 (dt, J= 15.2, 8.0 Hz, 2H),2.46 - 2.40 (m, 2H), 2.07 - 1.95 (m, 4 H, -CH₂-), 1.19 (dt, J = 14.0,7.1 Hz, 6 H, -CH₃).

Diethyl (4-(4-(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)butyl)-2-fluorobenzoyl)-L-glutamate (7h) Using thegeneral method for synthesis of compounds 6a-i, 5h (1.1 g, 3.46 mmol)was used to obtain 6h (0.25 g, 25%) as a white solid; TLC Rf= 0.0(MeOH:CHCl₃:HCl, 1:5:0.5); ¹H-NMR (400 MHz) (Me₂SO-d₆) δ 7.76 (t, J= 8.1Hz, 1 H, Ar), 7.23 (d, J = 2.9 Hz, 1 H, Ar), 7.16 - 7.05 (m, 2 H, Ar),6.03 (s, 2H, exch., 2-NH₂), 5.91 (d, J= 2.8 Hz, 1 H, Ar), 4.25 (t, J =6.8 Hz, 2 H, -CH₂-), 2.62 (t, J = 7.7 Hz, 2 H, -CH₂-), 1.72 (p, J= 6.9Hz, 2 H, -CH₂-), 1.48 (qd, J = 9.3, 8.8, 6.3 Hz, 2 H, -CH₂-). Thecompound was used for the next reaction without furthercharacterization. Using the general method for synthesis of compounds7a-i, 6h (0.15 g, 0.46 mmol) was used to obtain 7h (0.1 g, 43%) as abrown solid; TLC Rf= 0.23 (MeOH:CHCl₃:NH₄OH, 1:10:0.5); ¹H-NMR (400 MHz)(Me₂SO-d₆) ¹H NMR (400 MHz, DMSO-d₆) δ 7.88 (s, 1 H, exch., -NH), 7.55 -7.45 (m, 2 H, Ar), 7.17 - 7.06 (m, 2 H, Ar), 6.15 (d, J = 2.8 Hz, 1 H,Ar), 4.42 (s, 1 H, -CH), 4.29 (t, J = 6.8 Hz, 2 H, -CH₂-), 4.17 - 4.01(m, 4 H, -CH₂-), 4.05 - 3.93 (m, 2 H, -CH₂-), 3.66 (t, J= 12.4 Hz, 2 H,-CH₂-), 2.64 (t, J= 7.6 Hz, 2 H, -CH₂-), 1.74 (m, 2 H, -CH₂), 1.51 (d,J= 6.8 Hz, 2 H, -CH₂-), 1.26 - 1.13 (m, 6 H, -CH₃).

Diethyl (4-(5-(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)pentyl)-2-fluorobenzoyl)-L-glutamate (7i) Usingthe general method for synthesis of compounds 6a-i, 5i (1.2 g, 3.61mmol) was used to obtain 6i (0.32 g, 29%) as a white solid; TLC Rf= 0.0(MeOH:CHCl₃:HCl, 1:5:0.5); ¹H-NMR (400 MHz) (Me₂SO-d₆) δ 7.70 (td, J=8.2, 6.3 Hz, 1 H, Ar), 7.16 (d, J= 2.8 Hz, 1 H, Ar), 7.10 - 7.00 (m, 2H, Ar), 5.94 (s, 2 H, exch., -NH₂), 5.86 (d, J = 2.8 Hz, 1 H, Ar),4.23 - 4.14 (m, 2 H, -CH₂-), 2.60 (dt, J = 21.4, 7.8 Hz, 2 H, -CH₂-),1.75 -1.50 (m, 4 H, -CH₂-), 1.25 - 1.15 (m, 2H). The melting pointassessment suggested impurities and hence this compound was used for thenext reaction without further characterization. Using the general methodfor synthesis of compounds 7a-i, 6i (0.15 g, 0.44 mmol) was used toobtain 7i (0.11 g, 47.50%) as a grey solid TLC Rf= 0.23(MeOH:CHCl₃:NH₄OH, 1:10:0.5); ¹H-NMR (400 MHz) (Me₂SO-d₆) δ 11.17 (s, 1H, exch., -NH), 8.57 (dd, J = 7.5, 2.0 Hz, 1 H, exch., -NH), 7.50 (t, J= 7.8 Hz, 1 H, Ar), 7.38 (d, J = 2.9 Hz, 1 H, Ar), 7.16 - 7.07 (m, 2 H,Ar), 6.07 (d, J = 2.8 Hz, 1 H, Ar), 4.43 (ddd, J = 9.5, 7.4, 5.1 Hz, 1H, -CH), 4.24 (t, J = 7.1 Hz, 2 H, -CH₂-), 4.12 (qq, J = 7.0, 3.7 Hz, 2H, -CH₂-), 4.08 - 4.03 (m, 2 H, -CH₂-), 2.61 (t, J = 7.7 Hz, 2 H,-CH₂-), 2.46 - 2.40 (m, 2 H, -CH₂-), 2.09 (m, 2 H, -CH₂-), 1.75 (p, J =7.3 Hz, 2 H), 1.57 (q, J = 7.6 Hz, 2H), 1.22 - 1.17 (m, 8 H, -CH₂- and-CH₃).

(3-(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)propyl)benzoyl)-L-glutamicacid (AGF291) Using the general method for synthesis of targetcompounds, 7a (0.10 g, 0.2 mmol) was used to obtain AGF291 (0.06 g, 67%)as a white solid; TLC Rf=0.0 (MeOH:CHCl₃:HCl, 1:5:0.5); mp, 71.8-80.0°C.; ¹H-NMR (400 MHz) (Me₂SO-d₆) δ 12.10-11.20 (s, br, 3 H, exch., -COOHand -NH), 8.59 - 8.25 (m, 1 H, exch., -NH), 7.75 (d, J = 7.9 Hz, 2 H,Ar), 7.27 (d, J = 7.7 Hz, 2 H, Ar), 7.19 (d, J = 2.6 Hz, 1 H, Ar), 6.44(s, 2 H, exch., 2-NH₂), 5.89 (d, J = 2.7 Hz, 1 H, Ar), 4.32 - 4.19 (m, 3H, -CH and -CH₂), 2.57 (t, J = 7.3 Hz, 2 H, -CH₂-), 2.34 - 2.11 (m, 2 H,-CH₂-), 2.11 - 1.98 (m, 2 H, -CH₂-), 2.01-1.83 (m, 2 H, -CH₂-). Anal.Calcd. for C₂₁H₂₃ N₅O₆ 0.9 CH₃OH 0.8 HCl: C, 52.67; H, 5.53; N, 14.02;Found: C, 52.53; H, 5.63; N, 14.07.

(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)butyl)benzoyl)-L-glutamicacid (AGF300) Using the general method for synthesis of targetcompounds, 7b (0.10 g, 0.195 mmol) was used to obtain AGF300 (0.056 g,63%) as a white solid; TLC Rf = 0.0 (MeOH:CHCl₃:HCl, 1:5:0.5); mp,82.3-87.0° C.; 1H-NMR (400 MHz) (Me2SO-d6) δ 11.80-11.00 (s, br, exch.,3 H, COOH and NH), 8.26 (s, 1 H, exch., -NH), 7.71 (d, J = 7.9 Hz, 2 H,Ar), 7.33 - 7.09 (m, 3 H, Ar), 6.14 (s, 2 H, exch., 2-NH2), 5.86 (d, J =2.2 Hz, 1 H, Ar), 4.23 (m, 3 H, -CH- and -CH2-), 2.77 - 2.56 (m, 2 H,-CH₂-), 2.37 - 2.09 (m, 2 H, -CH₂-), 2.04 - 1.84 (m, 2 H, -CH₂-), 1.71(m, 2 H, -CH₂-), 1.46 (d, J = 7.4 Hz, 2 H, -CH₂). Anal. Calcd. forC₂₂H₂₅ N₅O₆ 0.77 HCl: C, 54.64; H, 5.37; N, 14.48. Found: C, 54.71; H,5.34; N, 14.28.

(5-(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)pentyl)benzoyl)-L-glutamicacid (AGF299) Using the general method for synthesis of targetcompounds, 7c (0.10 g, 0.195 mmol) was used to obtain AGF299 (0.050 g,56%) as a white solid; TLC Rf= 0.0 (MeOH:CHCl₃:HCl, 1:5:0.5); mp,82.3-84.8° C.; ¹H-NMR (400 MHz) (Me₂SO-d₆) δ 11.95 (s, 2 H, exch.,-COOH), 8.54 (d, J = 7.7 Hz, exch., -NH), 7.79 (d, J = 7.8 Hz, 2 H, Ar),7.40 - 7.00 (m, 3 H, Ar), 6.37 (s, 2 H, exch., 2-NH₂), 5.94 (d, J = 2.9Hz, 1 H, Ar), 4.38 (d, J = 8.2 Hz, 1 H, -CH-), 4.21 (t, J = 7.0 Hz, 2 H,-CH₂-), 2.60 (t, J = 7.7 Hz, 2 H, -CH₂-), 2.36 (t, J = 7.4 Hz, 2 H,-CH₂-), 2.17 - 1.82 (m, 2 H, -CH₂-), 1.80-1.65 (m, 2H, -CH₂-), 1.60-1.45(m, 2 H, -CH₂-), 1.26-1.00 (m, 2 H, -CH₂-). Anal. Calcd. C₂₃H₂₇N₅O₆ 1.08H₂O: C, 56.50; H, 6.01; N, 14.23. Found: C, 56.49; H, 5.83; N, 14.28.

(3-(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)propyl)thiophene-2-carbonyl)-L-glutamic acid (AGF331) Using the general methodfor synthesis of target compounds, 7d (0.10 g, 0.2 mmol) was used toobtain AGF331 (0.054 g, 61%) as a white solid; TLC Rf= 0.0(MeOH:CHCl₃:HCl, 1:5:0.5); mp, 150.3-154.3° C.; ¹H-NMR (400 MHz)(Me₂SOd₆) δ 12.20-11.10 (s, br, exch., 3 H, COOH and NH), 8.49 (d, J =7.8 Hz, 1 H, exch., -NH), 7.68 (d, J = 3.8 Hz, 1 H, Ar), 7.20 (d, J =2.8 Hz, 1 H, Ar), 6.90 (d, J = 3.8 Hz, 1 H, Ar), 5.91 (d, J = 2.8 Hz, 1H, Ar), 5.80 (s, 2 H, exch., 2-NH₂), 4.31 (dt, J = 28.9, 8.4 Hz, 3 H,-CH and -CH₂-), 2.72 (t, J = 7.7 Hz, 2 H, -CH₂-), 2.33 (t, J = 7.5 Hz, 2H, -CH₂-), 2.08 (dq, J = 12.7, 6.5, 5.7 Hz, 2 H, -CH₂-), 1.91 (m, 2 H,-CH₂-). Anal. Calcd. for C₁₉H₂₁ N₅O₅ S 0.8 H₂O: C, 49.41; H, 4.93; N,15.16; S, 6.94. Found: C, 49.44; H, 4.84; N, 15.13; S, 6.84.

(4-(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)butyl)thiophene-2-carbonyl)-L-glutamicacid (AGF318) Using the general method for synthesis of targetcompounds, 7e (0.10 g, 0.193 mmol) was used to obtain AGF318 (0.045 g,50%) as a white solid; mp, 148.3-150.2° C.; TLC Rf=0.0 (MeOH:CHCl₃:HCl,1:5:0.5); ¹H-NMR (400 MHz) (Me₂SO-d₆) δ 11.94 (s, 3 H, exch., -COOH andNH), 8.54 (d, J = 7.8 Hz, 1 H, exch., -NH), 7.69 (d, J = 7.8 Hz, 1 H,Ar), 7.33 (d, J = 3.8 Hz, 1 H, Ar), 6.96 (s, 2 H, exch., 2-NH₂), 6.85(d, J = 3.8 Hz, 1 H, Ar), 6.01 (d, J = 2.8 Hz, 1 H, Ar), 4.38 - 4.23 (m,3 H, -CH- and -CH₂-), 2.79 (t, J = 7.6 Hz, 2 H, -CH₂-), 2.34 (t, J = 7.4Hz, 2 H, -CH₂-), 2.14 - 1.92 (m, 2 H, -CH₂-), 1.76 (p, J = 6.9 Hz, 2 H,-CH₂-), 1.52 (p, J = 7.6 Hz, 2 H, -CH₂-). Anal. Calcd. for C₂₀H₂₃N₅O₆S0.58 HCl: C, 49.77; H, 4.92; N, 14.51; S, 6.64. Found: C, 49.80; H,5.08; N, 14.51; S, 6.74.

(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)pentyl)thiophene-2-carbonyl)-L-glutamicacid (AGF320) Using the general method for synthesis of targetcompounds, 7f (0.10 g, 0.188 mmol) was used to obtain AGF320 (0.072 g,81%) as a white solid; mp, 73.4-78.7° C.; TLC Rf=0.0 (MeOH:CHCl₃:HCl,1:5:0.5); ¹H-NMR (400 MHz) (Me₂SO-d₆) δ 12.32 (s, 2H, exch., -COOH),8.53 (d, J = 7.9 Hz, 1 H, exch., -NH), 7.69 (d, J = 3.8 Hz, 1 H, Ar),7.34 (d, J = 7.9 Hz, 1 H, Ar), 7.09 (s, 2H, exch., 2-NH₂), 6.87 (d, J =3.8 Hz, 1 H, Ar), 6.03 (d, J = 2.8 Hz, 1 H, Ar), 4.34 (d, J = 2.8 Hz, 1H, -CH), 4.23 (t, J = 7.0 Hz, 2 H, -CH₂-), 2.77 (t, J = 7.5 Hz, 2 H,-CH₂-), 2.34 (t, J = 7.5 Hz, 2 H, -CH₂-), 1.92 (d, J = 11.9 Hz, 2 H,-CH₂-), 1.74 (m, 2 H, -CH₂-), 1.61 (m, 2 H, -CH₂-), 1.25 (t, J = 7.5 Hz,2 H, -CH₂-). Anal. Calcd. for C₂₁H₂₅ N₅O₆ S 0.94 HCl: C, 49.48; H, 5.13;N, 13.74; S, 6.29. Found: C, 49.51; H, 5.21; N, 13.53; S, 6.31.

(3-(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)propyl)-2-fluorobenzoyl)-L-glutamicacid (AGF359) Using the general method for synthesis of targetcompounds, 7g (0.03 g, 0.07 mmol) was used to obtain AGF359 (0.04 g,67%) as a white solid; TLC Rf= 0.0 (MeOH:CHCl₃:HCl, 1:5:0.5); mp,175.8-183.1° C.; ¹H-NMR (400 MHz) (Me₂SOd₆) δ 12.10-11.20 (s, br, 3 H,exch., -COOH and -NH), 8.41 (dd, J = 7.6, 2.7 Hz, 1 H Ar), 7.53 (t, J=7.8 Hz, 1 H, Ar), 7.28 (d, J= 2.8 Hz, 1 H, Ar), 7.17 - 7.09 (m, 2H),6.50 (s, 2H), 5.98 (d, J= 2.8 Hz, 1H), 4.39 (ddd, J= 9.6, 7.6, 4.8 Hz,1H), 4.27 (t, J= 7.0 Hz, 2H), 2.61 - 2.54 (m, 2H), 2.40 - 2.31 (m, 2H),2.11 - 2.03 (m, 3H), 1.90 (dddd, J= 14.0, 9.5, 7.9, 6.3 Hz, 1H). MScalculated for C₂₁H₂₂FN₅O₆ [M+H]⁺ , 460.16. Found: 460.0. HPLC analysis:retention time, 14.42 min; peak area, 96.2%; eluent A, H₂O: eluent B,ACN; gradient elution (100% H₂O to 10% H₂O) over 45 min with flow rateof 0.3 mL/min and detection at 254 nm; column temperature, rt.

(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)butyl)-2-fluorobenzoyl)-L-glutamicacid (AGF347) Using the general method for synthesis of targetcompounds, 7h (0.40 g, 0.845 mmol) was used to obtain AGF347 (0.056 g,63%) as a white solid; TLC Rf = 0.0 (MeOH:CHCl3:HCl, 1:5:0.5); mp,161.6-162.4° C.; 1H-NMR (500 MHz) (Me2SO-d6) δ 8.43 (dd, J= 7.6, 2.6 Hz,1 H, exch., -NH), 7.51 (t, J= 7.7 Hz, 1 H, Ar), 7.23 (d, J= 2.9 Hz, 1 H,Ar), 7.13 - 7.05 (m, 2 H, Ar), 6.2 (s, 2H, exch., -NH₂), 5.92 (d, J= 2.8Hz, 1 H, Ar), 4.38 (ddd, J= 9.5, 7.5, 4.8 Hz, 1 H, -CH), 4.25 (t, J =6.8 Hz, 2 H, -CH₂-), 2.61 (t, J = 7.7 Hz, 2 H, -CH₂-), 2.40 - 2.28 (m, 2H, -CH₂-), 2.08 - 1.84 (m, 2 H, -CH₂-), 1.72 (p, J = 7.1 Hz, 2 H,-CH₂-), 1.48 (td, J = 8.5, 4.1 Hz, 2 H, -CH₂-). Anal. Calcd. forC₂₂H₂₄FN₅O₆ 0.58 HCl: C, 55.81; H, 5.11; N, 14.79; F,4.01 Found: C,53.30; H, 5.15; N, 14.18; F, 3.81.

(5-(2-amino-4-oxo-3,4-dihydro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)pentyl)-2-fluorobenzoyl)-L-glutamicacid (AGF355) Using the general method for synthesis of targetcompounds, 7i (0.05 g, 0.07 mmol) was used to obtain AGF355 (0.050 g,56%) as a grey solid; TLC Rf= 0.0 (MeOH:CHCl₃:HCl, 1:5:0.5); mp,138.5-145.7° C.; ¹H-NMR (400 MHz) (Me₂SOd₆) δ 8.42 (d, J = 9.0 Hz, 1 H,exch., -NH), 7.52 (d, J = 15.6 Hz, 1 H, Ar), 7.19 (d, J = 2.8 Hz, 1 H,Ar), 7.11 (d, J = 25.0 Hz, 2 H, Ar), 5.94 (s, 2 H, exch., -NH₂), 5.89(d, J = 2.8 Hz, 1 H, Ar), 4.39 (d, J = 21.9 Hz, 1 H, -CH), 4.20 (d, J =14.1 Hz, 2H, -CH₂-), 2.60 (d, J = 15.7 Hz, 2H, -CH₂-), 2.38 - 2.32 (m, 2H, -CH₂-), 2.12-1.91 (m, 2 H, -CH₂-), 1.73 (p, J = 7.2 Hz, 2 H, -CH₂-),1.57 (p, J= 7.7 Hz, 2 H, -CH₂-), 1.23 (d, J= 51.8 Hz, 2 H, -CH₂-). MScalculated for C₂₁H₂₂FN₅O₆ [M+H]⁺ , 488.19. Found: 487.9. HPLC analysis:retention time, 12.75 min; peak area, 95.23%; eluent A, H₂O: eluent B,ACN; gradient elution (100% H₂O to 10% H₂O) over 45 min with flow rateof 0.3 mL/min and detection at 254 nm; column temperature, rt.

Synthesis Schemes

For Formula l, Synthesis of AGF 323

a1) K2CO3, acetonitrile, 3-bromopropanol or 2.bromoethan-1-ol, reflux,24 h; a2) 3-aminipropanol, Cul, L-proline, dimethyl sulfoxide, r.t., 48h; b) PPh3, iodine, imidazole, DCM, 0oC, 1.5 h; c) ethyl3-amino-1H-pyrrole-2-carboxylate, NaH, DMF, 2 h, r.t.; d) (i)1,3-bis(methoxycarbonyl-2-methyl-2-thiopseudourea, MeOH, r.t. 16 h; (ii)NaOMe, MeOH, 16 h, r.t.; (iii) 1N NaOH, 55oC, 3 h; e) L-glutamic aciddiethyl ester hydrochloride, 2-chloro-4, 6-dimethoxy-triazine, NMM,anhydrous DMF, r.t., 12 h; f) 1N NaOH, r.t., 1 h; g) acetic anhydride,formic acid, 1 h, reflux, or acetic anhydride, rt, 12 h, ortrifluoracetic anhydride, rt, 4 h.

Synthesis of AGF323: Compound 11a was alkylated with 3-bromopropanol toafford alcohol 12a, which was subjected to the Appel reaction with PPh₃,imidazole and iodine to afford iodide 13a. Commercially available3-amino-1H-pyrrole-2-carboxylate was then alkylated with 13a (61%) andthe crude N-substituted pyrrole (14a) was directly subjected tocondensation with 1,3-bis(methoxycarbonyl)-2-methylthiopseudourea with 5equivalents of acetic acid as catalyst in MeOH. The hydrolysis of thecarbamate group formed, was carried out in situ with aqueous sodiumhydroxide at 55° C. to afford the 2-amino-4-oxo-pyrrolo[3,2-d]pyrimidine15a (14%). Conversion of free acid 15a to the corresponding L-glutamicacid diethyl esters 16a (38%) involved conventional peptide couplingwith L-glutamic acid diethyl ester hydrochloride using2-chloro-4,6-dimethoxy-1,3,5-triazine followed by chromatographicpurification. Hydrolysis of 16a with aqueous NaOH at room temperature,followed by acidification with 1 N HC1 afforded the target compoundAGF323 in 78% yields.

Additional Examples of Compounds of Formula 1:

-   1 n= 1, 3-4; R = H; X = CH₂; Ar = 1',4'-phenyl or 2',5'-thienyl-   2 n= 1-4; R = CH₃; X = CH₂; Ar = 1',4'-phenyl or 2',5'-thienyl-   3 n= 2-4; R = H; X = O, S, NH, NCHO, NCOCH₃, NCOCF₃; Ar =    1',4'-phenyl or 2',5'-thienyl-   4 n= 2-4; R = H; X = CH₂; Ar = 2'-fluoro-1',4'-phenyl-   AGF291 n = 2, Ar = 1',4'-phenyl, R = H, X = CH₂-   AGF299 n = 4, Ar = 1',4'-phenyl, R = H, X = CH₂-   AGF300 n = 3, Ar = 1',4'-phenyl, R = H, X = CH₂-   AGF307 n = 4, Ar = 1',4'-phenyl, R = CH₃, X = CH₂-   AGF312 n = 3, Ar = 1',4'-phenyl, R = CH₃, X = CH₂-   AGF318 n = 3, Ar = 2',5'-thienyl, R = H, X = CH₂-   AGF320 n = 4, Ar = 2',5'-thienyl, R = H, X = CH₂-   AGF323 n = 3, Ar = 1',4'-phenyl, R = H, X = O-   AGF331 n = 2, Ar = 2',5'-thienyl, R = H, X = CH₂-   AGF359 n = 2, Ar = 2'-fluoro-1',4'-phenyl, R = H, X = CH₂-   AGF347 n = 3, Ar = 2'-fluoro-1',4'-phenyl, R = H, X = CH₂-   AGF355 n = 4, Ar = 2'-fluoro-1',4'-phenyl, R = H, X = CH₂

For Formula I, Synthesis of AGF307, AGF312, AGF299, AGF359, AGF347 andAGF355:

a) PdCl2, PPh3, TEA, CuI, alcohol, acetonitrile, 1h, 100 °C, microwave;b) H2/Pd, high parr vessel, 24h, r.t.; c) (i) mesyl chloride, TEA, DCM,0 °C, 2 h; (ii) NaI, acetone, 4 h, reflux; d)ethyl-3-amino-1H-pyrrole-2-carboxylate or ethyl 3-amino-5-methyl-1H-pyrrole-2-carboxylate, NaH, DMF, 2h, r.t.; e) (i)1,3-bis(methoxycarbonyl)-2-methyl-2-thiopseudourea, MeOH, r.t., 16 h;(ii) NaOMe, MeOH, 16 h, r.t.; (iii) 1 N NaOH, 55 °C, 3 h; f) L-glutamicacid diethyl ester hydrochloride, 2-chloro- 4,6-dimethoxy-triazine, NMM,DMF, r.t., 12 h; g) 1N NaOH, r.t., 1 h

Synthesis of the target compounds AGF299, AGF307, AGF312, AGF359, AGF347and AGF355 started with a palladium-catalyzed Sonogashira coupling of4-iodobenzoate methyl ester or methyl 4-bromo-2-fluorobenzoate with theappropriate alkyne alcohols to afford the appropriate 4-substitutedalcohol benzoates 18 (78-88%). Catalytic hydrogenation afforded thesaturated alcohols 19 (85-98%). The alcohols were converted to themesylate derivatives using mesyl chloride and triethylamine base at 0°C. The mesylate derivatives were converted to their respective iodide 20(72-85%) using the Finkelstein reaction. The N-alkylation of iodides,using ethyl 3-amino-1H-pyrrole-2-carboxylate or ethyl3-amino-5-methyl-1H-pyrrole-2-carboxylate and sodium hydride underanhydrous conditions afforded the N-5 substituted pyrroles 21. The crudeNsubstituted pyrroles (21) were directly subjected to condensation with1,3-bis(methoxycarbonyl)-2-methylthiopseudourea with 5 equivalents ofacetic acid as catalyst and MeOH. The hydrolysis of the carbamate groupformed was carried out in situ with aqueous sodium hydroxide at 55° C.to afforded the 2-amino-4-oxo-pyrrolo[3,2-d]pyrimidines 22 (23- 30%).Conversion of free acids (22) to the corresponding L-glutamic aciddiethyl esters 23 (32- 75%) involved conventional peptide coupling withL-glutamic acid diethyl ester hydrochloride using2-chloro-4,6-dimethoxy-1,3,5-triazine followed by chromatographicpurification to afford the coupled products. Hydrolysis of 23 withaqueous NaOH at room temperature, followed by acidification with 1 N HC1in the cold, afforded target compounds AGF299, AGF307, AGF312, AGF347,AGF355 and AGF359 in 25-67% yield.

For Formula II, Synthesis of AGF287:

Reagents and conditions: (a) Pd(OAc)₂, LiCl, LiOAc, Bu₄NCl, DMF, 90° C.,2.5 h, 59%; (b) 5,5-dibromo-2,2-dimethyl-4,6-dioxo-1,3-dioxane 302, 1NHC1 in (Et)₂O, (Et)₂O, rt, 48 h; (c) CH₃COONa, MeOH, H₂O, 45° C., 4 h,20% yield over 2 steps; (d) (i) 1 N NaOH, rt, 12 h; (ii) 1 N HC1 , 61%yield; (e) NMM, CDMT, diethyl-L-glutamate, DMF, rt, 12 h, 81%; (f) (i)1N, NaOH, rt, 1 h; (ii) 0-4° C., 1 N HC1, 80%.

Synthesis of AGF287: Heck coupling reaction of commercially availablehex-5-en-1-ol 24 and methyl 4-bromo-2-fluorobenzoate 25 afforded theunsaturated, coupled alcohol that rearranged to the vinyl alcohol andtautomerized to afford the aldehyde 26 in 59% yield. Subsequentα-bromination of 26 with 5,5-dibromo-2,2-dimethyl-4,6-dioxo-1,3-dioxane(DBMA) at room temperature afforded corresponding α-bromo aldehyde 27,which was immediately condensed with 2,6-diamino-4-oxo-pyrimidine 28 inthe presence of sodium acetate to afford the 5-substitutedpyrrolo[2,3-d]pyrimidine 29 in 20% yield over 2 steps. The terminalester of 29 was subjected to base catalyzed hydrolysis to afford thepteroic acid 30 in 61% yield. The acid 30 was subsequently peptidecoupled with L-glutamate diethyl ester hydrochloride in the presence ofNMM and CDMT as the coupling agents to afford the diester 31 in 81%yield. Final saponification of the diesters with 1 N NaOH andneutralization to pH 4, provided target compound AGF287 in 80% yield.

For Formula III, Synthesis of AGF 315 and AGF 317

AGF315 n = 2, R = H, X = N, Y = CH, Z = CH₂

AGF317 n = 3, R = H, X = N, Y = CH, Z = CH₂

Synthesis of AGF315 and AGF317: Sonogashira coupling of 5-bromopicolinic L-glu ethyl ester (40) with corresponding alkyn-1-ol gavecompound 41 (scheme 5). Hydrogenation of triple bond of alkyne gavecompound 42. Oxidation of hydroxyl group of compound 42 using DMP gavealdehydes 43. Alpha bromination of aldehydes 43 with DMBA gave α-bromoaldehyde 44 which was used for next reaction without purification.Cyclization of α-bromo aldehyde 44 with 2,6-diamino-4-oxopyrimidine gavepyrrolo[2,3-d]pyrimidine 45. Hydrolysis of ester 45 produced finalcompounds AGF315 and AGF317.

Inhibitors of cytosolic C1 metabolism were among the first chemotherapyagents used for cancer and continue to be a mainstay for treating manycancers (8). However, the clinical utility of standard chemotherapydrugs is often limited by their toxicities toward normal tissues(reflecting a lack of tumor selectivity) and/or drug resistance.Discovery of new and potent inhibitors of tumor-selective pathways,while essential, remains a formidable challenge.

This invention provides compounds and pharmaceutically acceptable saltsthereof as novel inhibitors of C1 metabolism in mitochondria, theprimary catabolic pathway for serine and for synthesis of glycine (2-5).Serine catabolism in mitochondria is the principal source of C1 unitsfor cytosolic de novo purine and thymidylate biosynthesis, and ofreducing equivalents and ATP (2-5, 40). For SHMT2, the first enzyme inthe mitochondrial C1 pathway, CRISPR-Cas9 deletion in cultured cellsresults in defective mitochondrial respiration due to impaired synthesisof respiratory chain proteins (40, 41), accompanied by a commensurateincrease in glycolytic flux (40). We targeted SHMT2 with a novel seriesof 5-substituted pyrrolo[3,2-d]pyrimidine compounds of this invention,and we identify lead compounds of this invention, namely, AGF291, AGF320and AGF347 that inhibited proliferation of a broad spectrum of tumorsubtypes including lung (H460), colon (HCT116), and pancreatic (MIAPaCa-2) cancer.

We identified critical enzyme targets of our compounds throughglycine/nucleoside protection experiments and targeted metabolomics witha [2,3,3-²H]serine tracer, and identified SHMT2 in mitochondria as theprincipal intracellular target, along with SHMT1, and GARFTase and/orAICARFTase in the cytosol. Inhibition of all these intracellular targetswas confirmed by in vitro assays with purified recombinant enzymes. Ourfinding that SHMT1 is a direct target of our compounds resembles resultsfor a dual-SHMT1/SHMT2 pyrazolopyran inhibitor SHIN1 (21) and is ofparticular interest as this prevents metabolic “compensation” byreversal of SHMT1 catalysis in response to loss of SHMT2 activity (20,21).

Not to be bound by any particular theory, a number of pharmacodynamicfactors could contribute to the in vitro anti-tumor effects of the novelcompounds of this invention. These include transport across the plasmamembrane by PCFT and/or RFC and into mitochondria (potentially by themitochondrial folate transporter (11, 12)), metabolism to drugpolyglutamates, analogous to pemetrexed and other classic antifolates(6, 8), and binding to intracellular targets in both the mitochondriaand the cytosol. Variations in these parameters likely account fordifferences in relative anti-proliferative activities toward theassorted tumor models in this report. As compound of the presentinvention AGF291 showed selectivity for PCFT over RFC, this compound wasfurther tested in vivo with a MIA PaCa-2 xenograft model in SCID mice.AGF291 exhibited potent in vivo anti-tumor efficacy exceeding that ofGEM (standard-of-care for PaC), albeit at a 20-fold decreased dose.

The active 5-substituted pyrrolo[3,2-d]pyrimidine compounds describedherein expand upon earlier results with non-folate pyrazolopyraninhibitors of human SHMT2 (21) and, to our knowledge, represent thefirst bona fide inhibitors of this intracellular target withdemonstrated in vivo antitumor efficacy. Thus, inhibition of SHMT2,coupled with direct inhibition of multiple C1-depedent targets includingde novo purine biosynthesis and SHMT1, afford a valuable and excitingnew platform for future drug development for a variety of tumors.

The mouse work reported in this application was approved by the WayneState University Institutional Animal Care and Use Committee. Formetabolite measurements by targeted metabolomics, cultured cells wereincubated in media containing dialyzed fetal bovine serum and theisotopically labeled serine. For the 5-substitutedpyrrolo[3,2-d]pyrimidine compounds of this invention, complete chemicalsynthesis and compound characterizations are provided herein. These andall other experimental procedures are all described in detail herein.

References

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It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications that are within the spirit and scopeof the invention, as defined by the appended claims.

What is claimed is:
 1. A compound of Formula I, and optionally apharmaceutically acceptable salt thereof:

wherein, R is one selected from the group consisting of H and CH₃; n isan integer 4 when X is -CH₂- and Ar is 1,4-phenyl, or n is an integerranging from 1 to 4 when X is -CH₂- and Ar is either2'-fluoro-1,4-phenyl or 2,5-thienyl, or n is an integer ranging from 1to 4 when X is one selected from the group consisting of O, S, -NH-,-NHCHO-, -NHCOCH₃-, and -NHCOCF₃- and Ar is one selected from the groupconsisting of (a) 1,4-phenyl, (b) 2'-fluoro-1,4-phenyl, and (c)2,5-thienyl, or n is an integer 3 when X is -CH₂-, R is CH₃ and Ar is1,4-phenyl.
 2. A pharmaceutical composition comprising a therapeuticallyeffective amount of a compound of Formula I, and optionally apharmaceutically acceptable salt thereof:

wherein, R is one selected from the group consisting of H and CH₃; n isan integer 4 when X is -CH- and Ar is 1,4-phenyl, or n is an integerranging from 1 to 4 when X is -CH₂- and Ar is either2'-fluoro-1,4-phenyl or 2,5-thienyl, or n is an integer ranging from 1to 4 when X is one selected from the group consisting of O, S, -NH-,-NHCHO-, -NHCOCH₃-, and -NHCOCF₃- and Ar is one selected from the groupconsisting of (a) 1,4-phenyl, (b) 2'-fluoro-1,4-phenyl, and (c)2,5-thienyl, or n is an integer 3 when X is -CH₂-, R is CH₃ and Ar is1,4-phenyl.
 3. The pharmaceutical composition of claim 2 including apharmaceutically acceptable carrier.
 4. A compound of Formula II, andoptionally a pharmaceutically acceptable salt thereof:

wherein, R is one selected from the group consisting of H and CH₃; n isan integer ranging from 1 to 4; X is one selected from the groupconsisting of -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃- . 5.A pharmaceutical composition comprising a therapeutically effectiveamount of a compound of Formula II, and optionally a pharmaceuticallyacceptable salt thereof:

wherein, R is one selected from the group consisting of H and CH₃; n isan integer ranging from 1 to 4; X is one selected from the groupconsisting of -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃- . 6.The pharmaceutical composition of claim 5 including a pharmaceuticallyacceptable carrier.
 7. A compound of Formula III, and optionally apharmaceutically acceptable salt thereof:

wherein, R is one selected from the group consisting of H and CH₃; n isan integer ranging from 1 to 4; X is one selected from the groupconsisting of CH and N; Y is one selected from the group consisting ofCH and N; and Z is one selected from the group consisting of -CH₂- , O,S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃-.
 8. A pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundof Formula III, and optionally a pharmaceutically acceptable saltthereof:

wherein, R is one selected from the group consisting of H and CH₃; n isan integer ranging from 1 to 4; X is one selected from the groupconsisting of CH and N; Y is one selected from the group consisting ofCH and N; and Z is one selected from the group consisting of -CH₂- , O,S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃- .
 9. The pharmaceuticalcomposition of claim 8 including a pharmaceutically acceptable carrier.10. A method of treating a patient having cancer comprisingadministering a therapeutically effective amount of a compound ofFormula II, and optionally a pharmaceutically acceptable salt thereof:

wherein, R is one selected from the group consisting of H and CH₃; n isan integer ranging from 1 to 4; X is one selected from the groupconsisting of -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃- .11. A method of treating a patient having cancer comprisingadministering a therapeutically effective amount of a compound ofFormula III, and optionally a pharmaceutically acceptable salt thereof:

wherein, R is one selected from the group consisting of H and CH₃; n isan integer ranging from 1 to 4; X is one selected from the groupconsisting of CH and N; Y is one selected from the group consisting ofCH and N; and Z is one selected from the group consisting of -CH₂- , O,S, -NH-, -NHCHO-, -NHCOCH3-, and -NHCOCF₃-.
 12. A method of targetingmitochondrial metabolism comprising administering to a cancer patient aneffective amount of at least one compound selected from the group ofFormula I, and optionally a pharmaceutically acceptable salt thereof, ofFormula II, and optionally a pharmaceutically acceptable salt thereof,and of Formula III, and optionally a pharmaceutically acceptable saltthereof, of Formula III:

wherein, R is one selected from the group consisting of H and CH₃; n isan integer 4 when X is -CH₂- and Ar is 1,4-phenyl, or n is an integerranging from 1 to 4 when X is -CH₂- and Ar is either2'-fluoro-1,4-phenyl or 2,5-thienyl, or n is an integer ranging from 1to 4 when X is one selected from the group consisting of O, S, -NH-,-NHCHO-, -NHCOCH₃-, and -NHCOCF₃- and Ar is one selected from the groupconsisting of (a) 1,4-phenyl, (b) 2'-fluoro-1,4-phenyl, and (c)2,5-thienyl, or n is an integer 3 when X is -CH₂-, R is CH₃ and Ar is1,4-phenyl; and

wherein, R is one selected from the group consisting of H and CH₃; n isan integer ranging from 1 to 4; X is one selected from the groupconsisting of -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃- ;and

wherein R is one selected from the group consisting of H and CH₃; n isan integer ranging from 1 to 4; X is one selected from the groupconsisting of CH and N; Y is one selected from the group consisting ofCH and N; and Z is one selected from the group consisting of -CH₂- , O,S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃-.
 13. A method of targetingSHMT2 and MTHFD2 comprising administering to a cancer patient aneffective amount of at least one compound selected from the group ofFormula I, and optionally a pharmaceutically acceptable salt thereof, ofFormula II, and optionally a pharmaceutically acceptable salt thereof,and of Formula III, and optionally a pharmaceutically acceptable saltthereof, of Formula III:

wherein, R is one selected from the group consisting of H and CH₃; n isan integer 4 when X is -CH₂- and Ar is 1,4-phenyl, or n is an integerranging from 1 to 4 when X is -CH₂- and Ar is either2'-fluoro-1,4-phenyl or 2,5-thienyl, or n is an integer ranging from 1to 4 when X is one selected from the group consisting of O, S, -NH-,-NHCHO-, -NHCOCH₃-, and -NHCOCF₃- and Ar is one selected from the groupconsisting of (a) 1,4-phenyl, (b) 2'-fluoro-1,4-phenyl, and (c)2,5-thienyl, or n is an integer 3 when X is -CH₂-, R is CH₃ and Ar is1,4-phenyl; and

wherein, R is one selected from the group consisting of H and CH₃; n isan integer ranging from 1 to 4; X is one selected from the groupconsisting of -CH₂- , O, S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃- ;and

wherein R is one selected from the group consisting of H and CH₃; n isan integer ranging from 1 to 4; X is one selected from the groupconsisting of CH and N; Y is one selected from the group consisting ofCH and N; and Z is one selected from the group consisting of -CH₂-, O,S, -NH-, -NHCHO-, -NHCOCH₃-, and -NHCOCF₃- .
 14. The compound of claim 1wherein n=4, Ar is 1,4-phenyl, R is CH₃, and X is CH₂ and identified ascompound AGF307.
 15. The compound of claim 1 wherein n=3, Ar is2,5-thienyl, R is H, and X is CH₂ and identified as compound AGF318. 16.The compound of claim 1 wherein n=4, Ar is 2,5-thienyl, R is H, and X isCH₂ and identified as compound AGF320.
 17. The compound of claim 1wherein n=3, Ar is 1,4-phenyl, R is H, and X is O and identified ascompound AGF323.
 18. The compound of claim 1 wherein n=2, Ar is2,5-thienyl, R is H, and X is CH₂ and identified as compound AGF331. 19.The compound of claim 1 wherein n=4, Ar is 1,4-phenyl, R is H, and X isCH₂ and identified as compound AGF299.
 20. The compound of claim 1wherein n= 2, Ar is 2'-fluoro-1,4-phenyl, R is H, and X is CH₂ andidentified as compound AGF359.
 21. The compound of claim 1 wherein n= 3,Ar is 2'-fluoro-1,4-phenyl, R is H, and X is CH₂ and identified ascompound AGF347.
 22. The compound of claim 1 wherein n=4, Ar is2'-fluoro-1,4-phenyl, R is H, and X is CH₂ and identified as compoundAGF355.